Ceftolozane antibiotic compositions

ABSTRACT

This disclosure provides pharmaceutical compositions comprising ceftolozane, pharmaceutical compositions comprising ceftolozane and tazobactam, methods of preparing those compositions, and related methods and uses of these compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/856,075, filed Sep. 16, 2015, which claims the benefit of U.S.Provisional Application No. 62/051,859, filed Sep. 17, 2014 and is acontinuation-in-part application of U.S. application Ser. No.14/251,381, filed Apr. 11, 2014, which is a continuation application ofInternational Application No. PCT/US2014/028642, filed Mar. 14, 2014,which claims priority to U.S. Provisional Application No. 61/792,092,filed Mar. 15, 2013; U.S. Provisional Application No. 61/793,007, filedMar. 15, 2013; U.S. Provisional Application No. 61/882,936, filed Sep.26, 2013; and U.S. Provisional Application No. 61/893,436, filed Oct.21, 2013. The contents of these applications are incorporated hereby byreference in their entirety.

TECHNICAL FIELD

This disclosure relates to pharmaceutical compositions comprisingceftolozane, pharmaceutical compositions comprising tazobactam andceftolozane, methods of preparing those compositions, and relatedmethods and uses thereof.

BACKGROUND

Ceftolozane is a cephalosporin antibacterial agent. The antibacterialactivity of ceftolozane is believed to result from its interaction withpenicillin binding proteins (PBPs) to inhibit the biosynthesis of thebacterial cell wall which acts to stop bacterial replication.Ceftolozane is also referred to as “CXA-101”, FR264205,(6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadizol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate,or(6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylicacid). As used herein, the term “ceftolozane” means(6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylateor(6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylicacid in its free-base or salt form, including a sulfate form. Unlessotherwise indicated, the term “CXA-101” as used herein can refer toceftolozane in any pharmaceutically acceptable form, e.g., ceftolozanein its free-base or salt form, including a ceftolozane sulfate saltform. Ceftolozane sulfate is a pharmaceutically acceptable salt ofceftolozane that can be combined with sodium chloride and othercomponents to obtain an antibiotic composition suitable foradministration by injection or infusion.

Antibacterial pharmaceutical compositions can include ceftolozane as apharmaceutically acceptable salt formulated for intravenousadministration. Ceftolozane sulfate is a pharmaceutically acceptableceftolozane salt of formula (I) that can be formulated for intravenousadministration or infusion.

U.S. Pat. No. 7,129,232 discloses ceftolozane and various ceftolozanesalts. For example, a ceftolozane hydrogen sulfate salt is disclosedamong ceftolozane salts that can be formed “with a base or an acidaddition salt such as a salt with an inorganic base, for example, analkali metal salt [e.g., sodium salt, potassium salt, etc.], an alkalineearth metal salt [e.g., calcium salt, magnesium salt, etc.], an ammoniumsalt; a salt with an organic base, for example, an organic amine salt[e.g., trimethylamine salt, triethylamine salt, pyridine salt, picolinesalt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, etc.]; an inorganic acid additionsalt [e.g., hydrochloride, hydrobromide, sulfate, hydrogen sulfate,phosphate, etc.]; an organic carboxylic or sulfonic acid addition salt[e.g., formate, acetate, trifluoroacetate, maleate, tartrate, citrate,fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.];and a salt with a basic or acidic amino acid [e.g., arginine, asparticacid, glutamic acid, etc.].”

Antibiotic pharmaceutical compositions comprising a beta-lactamantibiotic compound (e.g., a cephalosporin) (i.e., an antibioticcompound possessing one or more beta-lactam moieties) can beadministered with a beta-lactamase inhibitor (BLI) compound. Forexample, beta-lactam antibiotic compounds such as ceftolozane or othercephalosporin antibiotic compounds can be formulated with, and/oradministered in combination with beta-lactamase inhibiting compounds(e.g., tazobactam and salts thereof) in order to mitigate the effects ofbacterial beta-lactamase enzymes that can lead to bacterial resistanceto antibiotic therapy. Tazobactam is a BLI compound approved for use infixed dose combination with piperacillin in an injectable antibacterialproduct available under commercial names ZOSYN (U.S.) and TAZOCIN (e.g.,in Canada, and the United Kingdom). Tazobactam sodium, a derivative ofthe penicillin nucleus, is a penicillanic acid sulfone having thechemical name sodium(2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-lazabicyclo[3.2.0]heptane-2-carboxylate-4,4-dioxide.The chemical formula is C₁₀H₁₁N₄NaO₅S and the molecular weight is 322.3.The chemical structure of tazobactam sodium is:

Ceftolozane can be formulated with tazobactam in antibiotic compositionscalled CXA-201 (ceftolozane/tazobactam for injection), comprisingceftolozane and tazobactam in a 2:1 weight ratio between the amount ofceftolozane active and the amount of tazobactam acid, regardless of thesalt forms of these compositions (e.g., 1,000 mg of ceftolozane activecan be included in about 1,147 mg of ceftolozane sulfate). CXA-201compositions include an amount of tazobactam in a pharmaceuticallyacceptable form providing 500 mg of tazobactam acid per 1,000 mg ofceftolozane active as acomposition formulated for injection, or forreconstitution prior to parenteral administration. In one productpresentation, CXA-201 can be provided in a single container comprisingceftolozane sulfate and tazobactam sodium, administered byreconstituting a container-unit dosage form container of solid CXA-201to form a reconstituted injectable formulation. In one presentation(e.g., for treatment of certain urinary tract infections and/or certainintr-abdominal infections), each unit dosage form container of CXA-201can contain 1000 mg of ceftolozane active (free base equivalent weight,e.g., provided as a pharmaceutically acceptable salt such as ceftolozanesulfate) and sterile tazobactam sodium at a quantity equivalent of 500mg of tazobactam free acid, in a solid form. In another presentation(e.g., for treatment of hospital acquired/ventilator-associatedbacterial pneumonia (HABP/VABP)), a CXA-201 product can include a unitdosage form container providing 2,000 mg of ceftolozane active (e.g., asan equivalent amount of ceftolozane sulfate) and 1,000 mg of tazobactamacid (e.g., as an equivalent amount of tazobactam sodium). CXA-201compositions display potent antibacterial activity against variousgram-negative infections such as, for example, complicatedintra-abdominal infection (cIAI), complicated urinary tract infection(cUTI), or hospital acquired/ventilator-associated bacterial pneumonia(HABP/VABP).

As disclosed herein, ceftolozane was initially found to be chemicallyunstable in certain lyophilized compositions evaluated during thedevelopment of CXA-101 and CXA-201 pharmaceutical compositions. Forexample, ceftolozane had a residual rate of about 51% in the absence ofa stabilizing agent during both a 3 day stability test at 70 degrees C.,indicating loss of almost half of the ceftolozane during the test(Example 2, Table 2A control sample), and a 5.88% reduction inceftolozane purity during a 7 day stability test at 60 degrees C. in theabsence of a stabilizing agent (Example 2, Table 2B control sample).Second, the formation of a number of additional ceftolozane degradationproducts formed during the preparation of initial compositions wasobserved by additional peaks using high performance liquidchromatography (HPLC) during stability tests of ceftolozane alone (e.g.,Peak P12 in Table 4 of Example 3, and the RT63′ peak in Table 15 ofExample 8), and testing of compositions with tazobactam and ceftolozaneformed by co-lyophilization of ceftolozane and tazobactam (e.g., RRT1.22 peak in Tables 12 and 13 of Example 7a). Accordingly, there remainsan unmet need to identify formulations and manufacturing methods thateffectively stabilize ceftolozane both in a solid and liquid form toprovide suitably stable pharmaceutical compositions comprisingceftolozane and tazobactam (both in a powder form for reconstitution andin a reconstituted form for parenteral delivery). These formulationsshould address the need to provide pharmaceutical compositions havingdesired levels of ceftolozane and tazobactam potency, as well as levelsof impurities that are therapeutically acceptable for parenteraladministration.

SUMMARY

As provided herein, ceftolozane can be stabilized in pharmaceuticalcomposition comprising ceftolozane and a stabilizing effective amount ofa stabilizing agent selected from the group consisting of: sodiumchloride, dextran 40, lactose, maltose, trehalose and sucrose. Thepharmaceutical compositions provided herein are based in part on thesurprising discovery that ceftolozane pharmaceutical compositionscomprising these stabilizing agents demonstrate improved ceftolozaneresidual rates (e.g., % ceftolozane remaining after 3 days at 70° C. asmeasured by HPLC) and/or chemical stability (e.g., lower reduction inceftolozane purity measured by HPLC after 7 days at 60° C. in astability test) compared control samples comprising ceftolozane withouta stabilizing agent.

Accordingly, preferred pharmaceutical antibiotic compositions caninclude ceftolozane sulfate and a stabilizing agent (e.g., 300 to 500 mgof a stabilizing agent per 1,000 mg ceftolozane active) in a lyophilizedunit dosage form (e.g., powder in a container). The unit dosage form canbe dissolved with a pharmaceutically acceptable carrier (e.g., 0.9%sodium chloride aqueous isotonic saline and/or water for injection), andthen intravenously administered. In certain ceftolozane compositions,the stabilizing agent can be selected from the group consisting of:sodium chloride, lactose, maltose and dextran 40, and/or selected fromthe group consisting of: sodium chloride, trehalose and sucrose.

In addition, the present disclosure provides ceftolozane pharmaceuticalcompositions based in part on the surprising discovery that ceftolozanepharmaceutical compositions comprising about 1000 mg of ceftolozaneactive per 189 mg sodium from sodium chloride demonstrate improvedchemical stability and purity compared with pharmaceutical compositionscomprising ceftolozane with comparatively less sodium chloride. Forexample, the invention is based in part on the discovery of the absenceof the “RT63 Impurity” (also referred to herein as “Formula III”) inHPLC analysis of pharmaceutical compositions comprising about 1,000 mgof ceftolozane and 189 mg sodium from sodium chloride. By comparison,reducing the amount of sodium chloride relative to ceftolozane in testedcompositions resulted in at least 1.5-fold greater impurity at RT=63minutes (observed by HPLC using the method described in Example 1). Theceftolozane formulations with reduced levels of sodium were not asstable as the ceftolozane formulation containing about 1,000 mg ofceftolozane per 189 mg sodium from sodium chloride per. Ceftolozaneformulations containing about 1,000 mg of ceftolozane effective perstabilizing-effective amount of sodium from sodium chloride maintainedthe level of RT63 Impurity below the detection limit (e.g., 0.03%)measured by HPLC using the method described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

In a further embodiment provided herein, ceftolozane sulfate isstabilized in pharmaceutical compositions by incorporation of aneffective amount of an inorganic salt stabilizing agent, in particular125 to 500 mg (e.g., 480 to 500 mg) of sodium chloride per gram ofceftolozane active. This is based in part on the surprising discoverythat ceftolozane pharmaceutical compositions comprising 125 to 500 mg(e.g., 480 to 500 mg) of sodium chloride per 1000 mg of ceftolozaneactive demonstrate improved ceftolozane purity and chemical stabilitycompared to pharmaceutical compositions comprising ceftolozane withcomparatively less sodium chloride. For example, the disclosedpharmaceutical compositions have an improved stability as a decrease inthe rate of ceftolozane purity and/or a decrease in the rate offormation of substances characterized by HPLC peaks 1 and 7 identifiedduring a 7-day stability study in Example 5. The disclosed ceftolozanepharmaceutical compositions comprise a stabilizing amount of sodiumchloride (e.g., 125 to 500 mg of sodium chloride [more specifically, 480to 500 mg] per 1000 mg of ceftolozane active). Certain preferredcompositions demonstrate improved ceftolozane purity (e.g., Table 6) andchemical stability (e.g., with respect to the composition of HPLC peak 1in Table 7) compared with pharmaceutical compositions comprisingceftolozane with comparatively less sodium chloride. For example, thedisclosed pharmaceutical compositions typically comprise less than about4% total impurity after being stored for seven days at 60° C., asdetermined by HPLC using the method described in Example 1.Alternatively, the disclosed pharmaceutical compositions comprise lessthan about 2% of the impurity represented by Peak 1 after being storedfor seven days at 60° C., as determined by HPLC using the methoddescribed in Example 1, where Peak 1 has a retention time relative toceftolozane of 0.1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

In an embodiment, pharmaceutical antibiotic compositions provided hereincan include ceftolozane sulfate and stabilizing amount of sodiumchloride (e.g., 125 to 500 mg more specifically 480 to 500 mg of sodiumchloride and 1,000 mg ceftolozane active) in a unit dosage form (e.g.,powder in a container). The unit dosage form can be dissolved with apharmaceutically acceptable carrier, and then intravenouslyadministered.

In another aspect, provided herein is a pharmaceutical compositioncomprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozaneactive, wherein the decrease in ceftolozane total purity is not greaterthan about 4% after storing the pharmaceutical composition for sevendays in a sealed container at 60° C., as determined by HPLC using themethod described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

In another aspect, provided herein is a pharmaceutical compositioncomprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozaneactive, wherein the increase in the amount of the impurity representedby Peak 1 is not greater than about 2% after storing the pharmaceuticalcomposition for seven days at 60° C., as determined by HPLC using themethod described in Example 1, where Peak 1 has a retention timerelative to ceftolozane of about 0.1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

In embodiments of these aspects, the pharmaceutical composition furthercomprises L-arginine, or citric acid. In other embodiments, thepharmaceutical composition is formulated for parenteral administration.In another embodiment, the compositions can be in a unit dosage formcomprising 125 mg to 500 mg sodium chloride, 1,000 mg of ceftolozane inthe form of ceftolozane sulfate, L-arginine and citric acid.

In other embodiments of these aspects, the pharmaceutical composition islyophilized. In another embodiment, the ceftolozane is ceftolozanesulfate.

In another aspect, provided herein is a unit dosage form injectablepharmaceutical composition comprising 125 mg to 500 mg sodium chlorideand 1,000 mg of ceftolozane active present as a composition of formula(I)

In another aspect, provided herein is a pharmaceutical compositioncomprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozaneactive present as ceftolozane sulfate, wherein the ceftolozane totalpurity is at least about 94% after storing the pharmaceuticalcomposition for three days at 60° C., as determined by HPLC using themethod described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

Applicants have further discovered pharmaceutical compositionscomprising ceftolozane and tazobactam with reduced and even undetectableamounts of the compound RRT 1.22, and methods of manufacturing thesecompositions. This is based in part on the discovery that the formationof RRT 1.22 can be reduced if not completely suppressed by lyophilizingceftolozane in the absence of tazobactam and then blending thelyophilized ceftolozane with a dry tazobactam composition, such as atazobactam composition lyophilized in the absence of ceftolozane (SeeExample 10 and the results reported in Tables 23 and 24). Based on theseresults, pharmaceutical compositions comprising ceftolozane andtazobactam, and pharmaceutical compositions prepared using ceftolozaneand tazobactam are provided herein. In particular, these pharmaceuticalcompositions can include ceftolozane and/or tazobactam with reduced oreven undetectable amounts of the compound RRT 1.22:

In one embodiment, a pharmaceutical composition can include ceftolozaneand tazobactam with less than 0.15%, 0.10%, 0.05% or 0.03% by weight; orfrom 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC or even undectableamounts of RRT 1.22 (e.g., less than about 0.03% of the compound RRT1.22 measured by HPLC). These pharmaceutical compositions can beobtained by a process comprising the steps of (a) lyophilizingceftolozane in the absence of tazobactam to obtain a lyophilizedceftolozane composition; and (b) combining the lyophilized ceftolozanewith tazobactam under conditions suitable to obtain said pharmaceuticalcomposition with the aforementioned purity levels. The combination ofthe lyophilized ceftolozane composition with tazobactam can includeblending the lyophilized ceftolozane composition with lyophilized orcrystalline tazobactam material.

Also provided herein is a pharmaceutical composition comprising a blendof separately lyophilized tazobactam and ceftolozane sulfate in anamount providing 1,000 mg of ceftolozane active per 500 mg of tazobactamactive, further comprising less than 0.15%, 0.10%, 0.05% or 0.03% byweight; from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC; or evenundectable amounts (e.g., less than about 0.03% by HPLC) of a compoundof formula (III) detectable at a retention time relative to ceftolozaneof 1.22 by high performance liquid chromatography (HPLC) using themethod of Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

CXA-201 compositions comprising less than about 0.15%, 0.10%, 0.05% or0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC ofthe compound of formula (III) can be obtained by a process comprisingthe steps of: (a) forming a first aqueous solution comprisingceftolozane (e.g., in a pharmaceutically acceptable salt such as formula(I)), (b) lyophilizing the first aqueous solution to obtain alyophilized ceftolozane composition, and (c) blending the lyophilizedceftolozane composition with a tazobactam composition (e.g., tazobactamacid lyophilized in the absence of ceftolozane) in an amount thatprovides a 2:1 weight ratio between the amount of ceftolozane active andtazobactam active.

In yet another aspect, provided herein is a method for the treatment ofa bacterial infection in a mammal, comprising administering to saidmammal a therapeutically effective amount of any one of thepharmaceutical compositions provided herein. In an embodiment, thebacterial infection is caused by the bacterial infection is caused bybacteria selected from the group consisting of: Staphylococcus aureus,Escherichia coli, Acinetobacter baumanii, Haemophilus influenzae,Klebsiella pneumonia, and Pseudomonas aeruginosa. In another embodiment,the bacterial infection is selected from the group consisting ofnosocomial pneumonia, complicated intra-abdominal infection andcomplicated urinary tract infection.

In yet another aspect, any of the pharmaceutical compositions providedherein may be used for the manufacture of a medicament for the treatmentof complicated intra-abdominal infection (cIAI), complicated urinarytract infection (cUTI), or hospital acquired/ventilator-associatedbacterial pneumonia (HABP/VABP).

In still another aspect provided herein, an antibiotic pharmaceuticalcomposition comprises ceftolozane (or a pharmaceutically acceptable saltthereof) and tazobactam (or a pharmaceutically acceptable salt thereof)in a fixed dose combination of 1,000 mg of ceftolozane active per 500 mgof tazobactam active, and a ceftolozane-stabilizing amount of 125 mg to500 mg sodium chloride per 1,000 mg of ceftolozane active.

In a further aspect disclosed herein, a pharmaceutical compositioncomprising stabilized ceftolozane sulfate is obtained by a processcomprising lyophilizing an aqueous solution comprising 125 mg to 500 mgsodium chloride with an amount of ceftolozane sulfate providing 1,000 mgof ceftolozane active, to obtain the lyophilized stabilized ceftolozanesulfate composition.

Yet another aspect provided herein discloses an antibacterialpharmaceutical composition comprising ceftolozane sulfate and tazobactamin a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactamactive, the pharmaceutical composition obtained by a process comprisingthe steps of:

a) lyophilizing a first aqueous solution in the absence of tazobactam,the first aqueous solution comprising ceftolozane sulfate prior tolyophilization to obtain a first lyophilized ceftolozane composition;and

b) blending the first lyophilized ceftolozane composition withtazobactam to obtain an antibacterial composition comprising less than0.13% by HPLC of a compound of formula (III) detectable at a retentiontime relative to ceftolozane of 1.22 by high performance liquidchromatography using the method described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25to detect:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart showing the steps for preparing a CXA-201composition comprising ceftolozane (referred to as CXA-101) andtazobactam using a blending process, wherein the ceftolozane andtazobactam are lyophilized separately prior to blending as describedherein.

FIG. 2 is a flowchart showing the steps for preparing a CXA-201composition comprising ceftolozane (referred to as CXA-101) andtazobactam using a co-lyophilization process, as described herein.

FIG. 3 is a plot of the data points from Table 6, showing the purity ofthe ceftolozane in CXA-101 compositions at 60° C. on day 0, day 1, day3, and day 7, as measured by HPLC, wherein the CXA-101 compositionscomprise ceftolozane and sodium chloride.

FIG. 4 is a plot of the data points from Table 7, showing the peak areaof the composition peak 1 in CXA-101 compositions at 60° C. on day 0,day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101compositions comprise ceftolozane and sodium chloride.

FIG. 5 is a plot of the data points from Table 8A, showing the totalpeak area of the composition with a RRT of 0.43 and the composition peak3 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7,as measured by HPLC, wherein the CXA-101 compositions compriseceftolozane and sodium chloride.

FIG. 6 is a plot of the data points from Table 8B, showing the peak areaof the composition peak 7 in CXA-101 compositions at 60° C. on day 0,day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101compositions comprise ceftolozane and sodium chloride.

FIG. 7 is a plot of the data points from Table 17, showing the purity ofceftolozane in CXA-201 compositions at 60° C. on day 0, day 1, day 3,and day 7, as measured by HPLC, wherein the CXA-201 compositionscomprise ceftolozane, tazobactam, and sodium chloride.

FIG. 8 is a plot of the data points from Table 18, showing the peak areaof the composition peak 1 in CXA-201 compositions at 60° C. on day 0,day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 9 is a plot of the data points from Table 19, showing the totalpeak area of the composition with a RRT of 0.43 and the composition peak3 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7,as measured by HPLC, wherein the CXA-201 compositions compriseceftolozane, tazobactam, and sodium chloride.

FIG. 10 is a plot of the data points from Table 20, showing the peakarea of the composition peak 7 in CXA-201 compositions at 60° C. on day0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 11 is a flowchart showing the manufacturing process for aceftolozane/tazobactam composition via co-filling.

FIG. 12 is a flowchart showing the process for preparing a CXA-201composition comprising ceftolozane (referred to as CXA-101) andtazobactam using a blending process in a dedicated production areaaccording to FDA Guidance.

FIG. 13 is a flowchart showing the process for preparing aceftolozane/tazobactam composition via co-filling in a dedicatedproduction area according to FDA Guidance.

FIG. 14 shows the mass spectra obtained for the RRT 1.22 compound.

FIG. 15 shows the chemical structures for certain peaks in the spectrain FIG. 14.

DETAILED DESCRIPTION

I. Stabilizing Ceftolozane

Ceftolozane can be stabilized in a pharmaceutical composition comprisingceftolozane and a stabilizing effective amount of a stabilizing agentselected from the group consisting of: sodium chloride, dextran 40,lactose, maltose, trehalose and sucrose. The stabilizing agent and thestabilizing effective amount of the stabilizing agent for combinationwith ceftolozane were determined by high performance liquidchromatography (HPLC) analysis, for example by detecting the ratio ofpeak areas obtained for ceftolozane compared to peaks for othersubstances.

Preferred stabilized ceftolozane compositions have a ceftolozaneresidual rate of greater than the residual rate measured for acomparable ceftolozane composition without the stabilizing agent. Unlessotherwise indicated, the residual rate is measured by detecting theamount of ceftolozane in a sample before and after a stability testusing HPLC, and determining the percentage of ceftolozane last duringthe stability test.

Referring to Example 2 (including Table 2A), the residual rate ofceftolozane in the control sample without a stabilizing agent (i.e., 100mg of ceftolozane) after 3 days at 70 degrees C. was 51.2%, meaning thatthe HPLC peak area after the stability test for ceftolozane was about51.2% of the HPLC peak area for ceftolozane at the start of thestability test (i.e., 3 days at 70 degrees C.). Sodium chloride, dextran40, lactose and maltose all showed higher ceftolozane residual ratesthan the control in Example 2, while ceftolozane was less stable thanthe control when combined with fructose, xylitol, sorbitol and glucose(e.g., as evidenced by a residual rate lower than that of the control).In one embodiment, stabilized ceftolozane compositions compriseceftolozane (e.g., ceftolozane sulfate) and a stabilizing effectiveamount of a stabilizing agent selected from the group consisting of:sodium chloride, dextran 40, lactose and maltose, where the stabilizingeffective amount provides a residual rate of at least 51.2% for theceftolozane in the stabilized ceftolozane composition after 3 days at 70degrees C. Preferably, the stabilized ceftolozane pharmaceuticalcompositions after 3 days at 70 degrees C. can comprise at least about70% of an initial amount of the stabilized ceftolozane in thepharmaceutical composition (i.e., a residual rate of about 70% orgreater, as shown in Example 2), where the % of ceftolozane is measuredby high performance liquid chromatography (HPLC) according to Example 1,i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

Referring to Example 2 (Table 2A), stabilized ceftolozane compositionsare characterized by a reduction in ceftolozane of less than about 5%after 7 days at 60 degrees C., where the % reduction of ceftolozane ismeasured by HPLC according to Example 1. The stabilized ceftolozanepharmaceutical composition comprising ceftolozane and a stabilizingagent selected from the group consisting of: sodium chloride, trehaloseand sucrose can lose less than 5% of the amount of ceftolozane after 7days at 60 degrees C., where the % loss of ceftolozane is measured byHPLC according to Example 1. Sodium chloride, trehalose and sucrose allshowed reduced reductions in ceftolozane purity after a 7 day stabilitytest at 60 degrees C. (as measured by the % HPLC peak corresponding toceftolozane before and after the stability test). In one embodiment,stabilized ceftolozane compositions comprise ceftolozane (e.g.,ceftolozane sulfate) and a stabilizing effective amount of a stabilizingagent selected from the group consisting of: sodium chloride, trehaloseand sucrose, where the stabilizing effective amount provides a reductionin ceftolozane purity of not more than about 5% (e.g, not more thanabout 4%) for the ceftolozane in the stabilized ceftolozane compositionafter 3 days at 70 degrees C.

Accordingly, in one aspect, provided herein is a pharmaceuticalcomposition comprising stabilized ceftolozane obtained by a processcomprising lyophilizing a composition including ceftolozane and astabilizing agent selected from the group consisting of: sodiumchloride, dextran 40, lactose, maltose, tehalose and sucrose, to obtaina lyophilized stabilized ceftolozane pharmaceutical composition. In anembodiment, the stabilizing agent is selected from the group consistingof: sodium chloride, trehalose and sucrose. In another aspect, providedherein is a pharmaceutical composition comprising stabilized ceftolozaneand a stabilizing agent selected from the group consisting of: sodiumchloride, dextran 40, lactose, maltose, tehalose and sucrose, whereinthe pharmaceutical composition after 3 days at 70 degrees C. comprisesat least about 70% of an initial amount of the stabilized ceftolozane inthe pharmaceutical composition.

In another aspect, provided herein is a container containing a unitdosage form of a pharmaceutical composition formulated for parenteraladministration for the treatment of complicated intra-abdominalinfections or complicated urinary tract infections, the pharmaceuticalcomposition comprising 1,000 mg of ceftolozane active, L-arginine,citric acid and about 300-500 mg of a stabilizing agent selected fromthe group consisting of: sodium chloride, trehalose, and sucrose,wherein the pharmaceutical composition after 3 days at 70 degrees C.comprises at least about 70% of an initial amount of the ceftolozaneactive in the pharmaceutical composition.

Various ceftolozane compositions are described herein. One stabilizedceftolozane composition comprises ceftolozane (e.g., ceftolozanesulfate), L-arginine, citric acid, and a stabilizing agent. Preferably,the stabilized ceftolozane composition comprises 1,000 mg of ceftolozaneactive, L-arginine and stabilizing-effective amount of the stabilizingagent. The stabilizing effective amount can be readily determined usingHPLC and a stability test as disclosed herein. The stabilizing-effectiveamount can be effective to provide: (1) a residual rate measured by HPLCof ceftolozane of at least about 51.2% (including, e.g., at least about70%, and at least about 80%) after 3 days at 70 degrees C. and/or (2) areduction in ceftolozane purity measured by HPLC of not more than about5.11% (including, e.g., reductions of not more than about 5%, or 4%)after 7 days at 60 degrees C. Examples of stabilizing effective amountsinclude 100 mg-500 mg of the stabilizing agent per 1,000 mg of theceftolozane active, more preferably about 300-500 mg of the stabilizingagent per 1,000 mg of the ceftolozane active.

In the screening of ceftolozane stabilizing agents, it has been foundthat, surprisingly, a preferred amount of sodium chloride can improvethe stability of ceftolozane, including ceftolozane in the ceftolozanesulfate form. For example, in one experiment, a ceftolozane compositioncomprising about 100 mg (about 1.71 mmol) sodium chloride per 100 mg(about 0.15 mmol) of ceftolozane was more stable compared to manyceftolozane compositions comprising known stabilizing sugars, such asfructose, xylitol, sorbitol, glucose, and D-mannitol, and as stable asother ceftolozane compositions comprising the same amount of certainsugars, such as dextran 40, lactose, and maltose (see Example 2).Interestingly, additional experiments demonstrated that the use ofmaltose in a ceftolozane composition resulted in a significant amount ofadditional compounds (see Example 3).

Surprisingly, pharmaceutical compositions comprising ceftolozane and 125to 1000 mg sodium chloride per 1000 mg of ceftolozane have been observedto exhibit better chemical stability over the course of time and/or inthe presence of heat, and fewer additional compounds than thosepharmaceutical compositions comprising ceftolozane and less sodiumchloride (i.e., less than 125 mg sodium chloride per 1000 mg ofceftolozane) (see, e.g., Example 5). In particular embodiments describedherein, the pharmaceutical compositions comprising ceftolozane and 125to 500 mg sodium chloride per 1000 mg of ceftolozane have been found tobe more stable than the compositions comprising ceftolozane and lessthan 125 mg sodium chloride per 1000 mg of ceftolozane.

Ceftolozane compositions having 50-481 mg of sodium chloride per 1,000mg ceftolozane active were prepared as described in Table 5 and testedfor stability as described in Example 5. Ceftolozane was more stable incompositions containing at least 125 mg of sodium chloride per 1,000 mgof ceftolozane active, as measured by high performance liquidchromatography (HPLC) analysis by detecting the ratio of peak areasobtained for ceftolozane compared to peaks for other substances. Unlessotherwise indicated, HPLC measurements reported herein are obtainedusing the conditions described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

During the stability test of Example 5, ceftolozane samples containing125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozaneactive showed a decrease in ceftolozane total purity measured by HPLCthat was at least about 35% less than reductions in ceftolozane totalpurity observed for formulations containing 50 mg or 75 mg sodiumchloride per 1,000 mg ceftolozane active. Thus, ceftolozane compositionshaving at least 125 mg or more sodium chloride relative to the fixedamount of ceftolozane were about 35-90% more stable than comparableceftolozane compositions having less than 125 mg sodium chloride (e.g.,the % decrease in ceftolozane for the sample containing 75 mg sodiumchloride was about 35% greater than the comparable % decrease inceftolozane for the sample containing 190 mg sodium chloride). Inaddition, samples obtained from ceftolozane compositions containing 125mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozaneactive showed a decrease in ceftolozane that was up to about 90% lessthan reductions in ceftolozane observed for formulations containing 50mg or 75 mg sodium chloride per (e.g., the % decrease in ceftolozane forthe sample containing 50 mg sodium chloride was about 90% greater thanthe comparable % decrease in ceftolozane for the sample containing 481mg sodium chloride).

The ceftolozane sodium-stabilized compositions having 125 mg or moresodium chloride relative to the fixed amount of 1,000 mg ceftolozaneactive also had lower quantities of additional substances identified bypeaks 1 and 7 having characteristic retention times measured by HPLC(see Table 1, indicating retention times of about 0.1 for peak 1 andabout 1.3 for peak 7 relative to ceftolozane measured according to theHPLC method of Example 1). In particular, these sodium chloridestabilized ceftolozane compositions were characterized by about 37-94%less of the material of peak 1 and about 38-306% less of the material ofpeak 7 (measured by corresponding HPLC peak areas) than comparableceftolozane compositions having less than 125 mg sodium chloride (e.g.,see 7-day stability study in Example 5). Referring to the data in Table7 (FIG. 4), the amount of the composition of peak 1 (measured by HPLCaccording to Example 1) was measured by the % increase in the peak 1HPLC peak during the 7-day stability test of Example 5.

In particular, samples containing 125 mg, 190 mg and 481 mg of sodiumchloride per 1,000 mg of ceftolozane active showed at least a 37%reduction in the amount of the peak 1 composition observed for theseformulations containing at least 125 mg sodium chloride per 1,000 mgceftolozane active, compared to the compositions with 50 mg or 75 mgsodium chloride per 1,000 mg of ceftolozane active (e.g., the % increasein peak 1 for the sample containing 75 mg sodium chloride was about 37%greater than the comparable % decrease in ceftolozane for the samplecontaining 190 mg sodium chloride). In addition, compositions containing125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozaneactive showed up to a 94% reduction in the amount of the peak 1composition observed for these formulations containing at least 125 mgsodium chloride per 1,000 mg ceftolozane active, compared to thecompositions with 50 mg or 75 mg sodium chloride per 1,000 mg ofceftolozane active (e.g., the % increase in peak 1 for the samplecontaining 50 mg sodium chloride was about 94% greater than thecomparable % decrease in ceftolozane for the sample containing 481 mgsodium chloride).

The formulation of pharmaceutical compositions can be selected tominimize decomposition of the constituent drug substances and to producea composition that is stable under a variety of storage conditions.

Provided herein are pharmaceutical compositions useful for the treatmentof bacterial infections comprising ceftolozane and sodium chloride,wherein the sodium chloride is present in an amount sufficient tostabilize the ceftolozane. Also provided herein are pharmaceuticalcompositions comprising ceftolozane, tazobactam, and sodium chloride,wherein the sodium chloride is present in an amount sufficient tostabilize the ceftolozane. Advantageously, these pharmaceuticalcompositions have fewer additional compounds and are more chemicallystable, and can therefore be stored for longer periods of time.

In one embodiment, provided herein is a pharmaceutical compositioncomprising ceftolozane and 125 mg sodium chloride per 1000 mg ofceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg ofceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane,300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodiumchloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg ofceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane,wherein the purity of the ceftolozane in the composition is 75% orgreater after 3 days at 70° C. In another embodiment, provided herein isa pharmaceutical composition comprising ceftolozane and about 487 mgsodium chloride per 1000 mg of ceftolozane, wherein the purity of theceftolozane in the composition is 75% or greater after 3 days at 70° C.In certain embodiments, the purity of the ceftolozane in the compositionis 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97%or greater, or 99% or greater after 3 days at 70° C.

In another embodiment, provided herein is a pharmaceutical compositioncomprising ceftolozane and 125 mg sodium chloride per 1000 mg ofceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg ofceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane,300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodiumchloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg ofceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane,wherein the purity of the ceftolozane in the composition is 94.8% orgreater after 3 days at 60° C. In another embodiment, provided herein isa pharmaceutical composition comprising ceftolozane and about 487 mgsodium chloride per 1000 mg of ceftolozane, wherein the purity of theceftolozane in the composition is 94.8% or greater after 3 days at 60°C. In certain embodiments, the purity of the ceftolozane in thecomposition is 95% or greater, 96% or greater, 97% or greater, 98% orgreater, or 99% or greater after 3 days at 60° C.

In still another embodiment, provided herein is a pharmaceuticalcomposition comprising ceftolozane and 125 mg sodium chloride per 1000mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg ofceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane,300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodiumchloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg ofceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane,wherein the purity of the ceftolozane in the composition decreases by3.1% or less after 3 days at 60° C. In another embodiment, providedherein is a pharmaceutical composition comprising ceftolozane and about487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity ofthe ceftolozane in the composition decreases by 3.1% or less after 3days at 60° C. In certain embodiments, the purity of the ceftolozane inthe composition decreases by 3.0% or less, 2.5% or less, 2.0% or less,1.5% or less, or 1% or less after 3 days at 60° C.

In another aspect, provided herein is a pharmaceutical compositioncomprising about 1,000 mg of ceftolozane active per 189 mg sodium fromsodium chloride, and not more than 0.03% by high performance liquidchromatography (HPLC) of a RT63 Impurity at a retention time of about 63minutes observed by HPLC using the method described in Example 1, thepharmaceutical composition obtained by a process comprising the step oflyophilizing an aqueous solution comprising 189 mg sodium from sodiumchloride per 1,000 mg of ceftolozane active in the form of ceftolozanesulfate to obtain a lyophilized ceftolozane composition, and formulatingthe pharmaceutical composition from the lyophilized ceftolozanecomposition.

In one embodiment, the pharmaceutical composition comprises a total of1,000 mg of ceftolozane active. In another aspect, provided herein is apharmaceutical composition obtained by a process comprising the step oflyophilizing an aqueous solution comprising 189 mg sodium from sodiumchloride per 1,000 mg of ceftolozane in the form of ceftolozane sulfateto obtain a lyophilized ceftolozane composition.

In one embodiment, the pH of the aqueous solution is 5.0 to 7.0, e.g.,6.0 to 7.0, and the aqueous solution further comprises L-arginine. Inanother embodiment, the pharmaceutical composition is formulated forparenteral administration and further comprises citric acid. In anotherembodiment, the composition is a unit dosage form in a containercomprising tazobactam and 189 mg sodium from sodium chloride per 1,000mg of ceftolozane active in the form of ceftolozane sulfate.

In another embodiment, the aqueous solution further comprises L-arginineand citric acid; the pH of the aqueous solution is 6.0 to 7.0 prior tolyophilization; and the pharmaceutical composition further comprisestazobactam blended with the lyophilized ceftolozane composition.

In still another aspect, provided herein is a container containing aunit dosage form of a pharmaceutical composition formulated forparenteral administration for the treatment of complicatedintra-abdominal infections or complicated urinary tract infections, thepharmaceutical composition comprising 189 mg sodium from sodiumchloride, and 1,000 mg ceftolozane active in the form of ceftolozanesulfate.

In one embodiment, the container comprises the ceftolozane sulfate,tazobactam and the sodium chloride and not more than 0.03% by highperformance liquid chromatography (HPLC) of a RT63 Impurity at aretention time of about 63 minutes observed by HPLC using the methoddescribed in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

Typically, antibiotic compositions do not contain sodium chloride orcontain only a small amount of sodium chloride. For example, MAXIPIME®,which is approved for pneumonia, empiric therapy for febrile neutropenicpatients, uncomplicated and complicated urinary tract infections,uncomplicated skin and skin structure infections, and complicatedintra-abdominal infections, is a dry mixture of cefepime hydrochlorideand L-arginine, wherein the mixture does not contain sodium chloride.CEFAZOLIN® for injection, which is approved for respiratory tractinfections, urinary tract infections, skin and skin structureinfections, biliary tract infections, bone and joint infections, genitalinfections, septicemia, and endocarditis and perioperative prophylaxis,comprises lyophilized cefazolin sodium that does not contain additionalsodium salt. Furthermore, ROCEPHIN®, which is approved for lowerrespiratory tract infections, acute bacterial otitis media, skin andskin structure infections, urinary tract infections, uncomplicatedgonorrhea, pelvic inflammatory disease, bacterial septicemia, bone andjoint infections, intra-abdominal infections, meningitis, and surgicalprophylaxis, comprises ceftriaxone sodium that only comprises 13.5 mg offree sodium per 1000 mg of ceftriaxone sodium, which equals about 34 mgsodium chloride per 1000 mg of ceftriaxone sodium if the free sodium isin sodium chloride form. In contrast, the pharmaceutical compositionsprovided herein (compositions comprising ceftolozane and sodiumchloride, and compositions comprising ceftolozane, tazobactam, andsodium chloride), have high amounts of sodium chloride, e.g., 125-1000mg sodium chloride per 1000 mg of ceftolozane.

Ceftolozane

The compound5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-2-{[(6R,7R)-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}-1-methyl-1H-pyrazoliummonosulfate (also known also as ceftolozane sulfate, FR264205,“CXA-101”) is a cephalosporin compound (shown below), the synthesis ofwhich is described in U.S. Pat. No. 7,129,232, wherein the compound isalso named7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(1-carboxy-1-methylethoxyimino)acetamido]-3-{3-amino-4-[3-(2-aminoethyl)ureido]-2-methyl-1-pyrazolio}methyl-3-cephem-4-carboxylate.Ceftolozane has the chemical formula below and CAS registry number689293-68-3. “Ceftolozane” can be provided as the salt, ceftolozanesulfate.

Unless otherwise indicated herein, the phrase “1000 mg ceftolozane” or“1 g ceftolozane” refers to an amount of ceftolozane containing the freebase equivalent weight of ceftolozane provided in the free base form orany suitable salt form, as appropriate. For example, a compositioncontaining 1000 mg of ceftolozane in the ceftolozane sulfate solid formwill include greater than 1000 mg of material (e.g., due to at least theadditional weight of the sulfate counter ion). Preferably, theceftolozane is present as ceftolozane sulfate. If a ceftolozane sulfatecomposition contains “1000 mg of ceftolozane” then it includes an amountof ceftolozane sulfate comprising 1000 mg of the ceftolozane molecule infree base equivalent form. For example, as shown in Table 29, 1147 mgceftolozane sulfate corresponds to 1000 mg of ceftolozane free base.

In another embodiment, “1000 mg ceftolozane” refers to an amount ofceftolozane that is considered a bioequivalent by the United States Foodand Drug Administration (FDA), i.e. for which 90% CI of the relativemean Cmax, AUC(0-t) and AUC(0-∞) is within 80.00% to 125.00% of thereference formulation in the fasting state (see: “Guidance for Industry:Bioavailability and Bioequivalence Studies for Orally Administered DrugProducts—General Considerations”. Center for Drug Evaluation andResearch, United States Food and Drug Administration, 2003).

“Ceftolozane active” refers to the active portion of a salt form ofceftolozane, i.e., the free base form of ceftolozane.

As used herein, “125 to 1000 mg sodium chloride per 1000 mg ofceftolozane” refers to a ratio of sodium chloride to ceftolozane freebase equivalent. For example, “125 to 1000 mg sodium chloride per 1000mg of ceftolozane” includes, for example, 62.5 to 500 mg sodium chlorideper 500 mg of ceftolozane, as well as, for example, 25 to 200 mg sodiumchloride per 200 mg ceftolozane, etc.

In another aspect, provided herein is a method for the treatment ofbacterial infections in a mammal, comprising administering to saidmammal a therapeutically effective amount of a pharmaceuticalcomposition comprising ceftolozane as described herein.

II. Ceftolozane in the Presence of Tazobactam

It has also been observed that pharmaceutical compositions comprisingceftolozane, tazobactam, and 125 to 1000 mg sodium chloride per gram ofceftolozane exhibit better chemical stability and fewer additionalcompounds than those pharmaceutical compositions comprising ceftolozaneand tazobactam, but less sodium chloride (see, e.g., Example 8). Inparticular embodiments described herein, the pharmaceutical compositionscomprising ceftolozane, tazobactam, and 125 to 500 mg sodium chlorideper 1000 mg of ceftolozane have been found to be more stable than thecompositions comprising ceftolozane, tazobactam, and less than 125 mgsodium chloride per gram of ceftolozane.

Adding high amounts of sodium chloride to CXA-201 compositions (e.g.,125-1000 mg sodium chloride per 1000 mg of ceftolozane, 125-500 mgsodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chlorideper 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg ofceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane,450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodiumchloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per1000 mg of ceftolozane) also inhibits the formation of certainadditional compounds. Adding about 487 mg sodium chloride per 1000 mg ofceftolozane to CXA-201 composition can also inhibit the formation ofcertain additional compounds. For example, in one experiment, CXA-201compositions comprising 125-481 mg sodium chloride per 1000 mgceftolozane developed a reduced amount of a composition having aretention time of 63 minutes (“RT 63”) after three months at 25° C. (seethe HPLC measurements shown in Example 8A).

Accordingly, in one aspect, provided herein is a pharmaceuticalcomposition comprising ceftolozane, tazobactam, and 125-1000 mg sodiumchloride per 1000 mg of ceftolozane, e.g., 125-500 mg sodium chlorideper 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg ofceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane,400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodiumchloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg ofceftolozane. In another embodiment, provided herein is a pharmaceuticalcomposition comprising ceftolozane, tazobactam, and about 487 mg sodiumchloride per 1000 mg of ceftolozane.

In another embodiment, provided herein is a pharmaceutical compositioncomprising ceftolozane, tazobactam, and 125-1000 mg sodium chloride per1000 mg of ceftolozane, e.g., 125-500 mg sodium chloride per 1000 mg ofceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane,300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodiumchloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg ofceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane,wherein the purity of the ceftolozane in the composition is 94.9% orgreater after 3 days at 60° C. In another embodiment, provided herein isa pharmaceutical composition comprising ceftolozane, tazobactam, andabout 487 mg sodium chloride per 1000 mg of ceftolozane, wherein thepurity of the ceftolozane in the composition is 94.9% or greater after 3days at 60° C. In certain embodiments, the purity of the ceftolozane inthe composition is 95% or greater, 96% or greater, 97% or greater, 98%or greater, or 99% or greater after 3 days at 60° C.

Tazobactam

The compound(2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid 4,4-dioxide (also known as tazobactam) is a β-lactamase inhibitorof the following structure:

As used herein, tazobactam can be a free acid, a sodium salt, anarginine salt, or a hydrate or solvate thereof. The phrases “250-750 mgtazobactam”, “250-700 mg tazobactam,” “300-700 mg tazobactam”, “300-650mg tazobactam”, “350-650 mg tazobactam”, “350-600 mg tazobactam”,“400-600 mg tazobactam”, “400-550 mg tazobactam”, “450-550 mgtazobactam” or “about 500 mg tazobactam” refer to an amount oftazobactam containing the free acid equivalent weight of tazobactamprovided in the free acid form or any suitable salt form. For example, acomposition containing 500 mg of tazobactam in the tazobactam sodiumsolid form will include greater than 500 mg of material (e.g., due to atleast the additional weight of the sodium counter ion). For example, asshown in Table 29, 537 mg tazobactam sodium corresponds to 500 mg oftazobactam free acid. Preferably, the tazobactam is present astazobactam sodium. If a tazobactam sodium composition contains “500 mgof tazobactam” then it includes an amount of tazobactam sodiumcomprising 500 mg of the tazobactam molecule in free acid equivalentform.

In an embodiment, the tazobactam is tazobactam sodium sterile powder. Inyet a further embodiment, the tazobactam sodium sterile powder isgenerated by neutralizing tazobactam acid with sodium bicarbonatefollowed by lyophilization.

As used herein, the term “tazobactam active” refers to the activeportion of a salt form of tazobactam, i.e., tazobactam free acid.

In certain embodiments, the pharmaceutical compositions further comprisetazobactam sodium at a quantity equivalent of 500 mg of tazobactam freeacid in a lyophilized powder form per 1000 mg of ceftolozane (anhydrous,free base equivalent).

III. Liquid Pharmaceutical Compositions or Formulations

In another aspect, provided herein is a liquid pharmaceuticalcomposition (e.g., an intravenous infusion solution) comprisingceftolozane and tazobactam, wherein the composition is suitable forintravenous administration. In one embodiment, the composition furthercomprises 125-1000 mg sodium chloride per 1000 mg of ceftolozane. Inanother embodiment, the composition further comprises 125-500 mg sodiumchloride per 1000 mg of ceftolozane. In an embodiment, the liquidpharmaceutical composition (e.g., an intravenous infusion solution) isprepared by reconstitution of a ceftolozane and tazobactam compositionwith sterile water and/or normal sterile saline, followed by dilutionwith sterile water and/or normal sterile saline. In an embodiment, theliquid pharmaceutical composition (e.g., an intravenous infusionsolution) is prepared by reconstitution of a ceftolozane and tazobactamcomposition with normal sterile saline, followed by dilution with normalsterile saline. In another embodiment, the liquid pharmaceuticalcomposition (e.g., an intravenous infusion solution) has an osmolalitybetween about 300 mOsm/kg and 900 mOsm/kg, including injectableformulations with an osmolality of 350-900 mOsm/kg to 350-800 mOsm/kg,400-500 mOsm/kg and 500-600 mOsm/kg. In a further embodiment, the liquidpharmaceutical composition (e.g., an intravenous infusion solution)comprising 1,000 mg ceftolozane active and 500 mg of tazobactam active(as pharmaceutically acceptable salts thereof) has an osmolality that isbetween about 400 mOsm/kg and 500 mOsm/kg (e.g, 446-478 mOsm/kg, 440-480mOsm/kg, 420-490 mOsm/kg). In a further embodiment, the liquidpharmaceutical composition (e.g., an intravenous infusion solution)comprising 2,000 mg ceftolozane active and 1000 mg of tazobactam active(as pharmaceutically acceptable salts thereof) has an osmolality that isbetween about 500 mOsm/kg and 650 mOsm/kg. In yet a further embodiment,the liquid pharmaceutical composition (e.g., an intravenous infusionsolution) has an osmolality that is less than about 600 mOsm/kg (e.g,290-610 mOsm/kg, 350-605 mOsm/kg, 550-605 mOsm/kg, 589-604 mOsm/kg). Inanother embodiment, the ceftolozane and tazobactam of the liquidpharmaceutical composition (e.g., an intravenous infusion solution) arecontrolled to pH 5 to 7. In a further embodiment, the ceftolozane andtazobactam of the liquid pharmaceutical composition (e.g., anintravenous infusion solution) are controlled to about pH 6.

In one embodiment, the methods further comprise reconstituting thelyophilized mixture in an aqueous solvent, such that the resultingsolution is suitable for infusion. The mixture can be reconstituted insaline and/or sterile water for injection.

Methods of Preparing Pharmaceutical Compositions Comprising Ceftolozaneand Sodium Chloride

Pharmaceutical compositions comprising ceftolozane andstabilizing-effective amount of a stabilizing agent can be obtained bylyophilization. As is known to those skilled in the art, lyophilizationis a process of freeze-drying in which water is sublimed from a frozensolution of one or more solutes. Specific methods of lyophilization aredescribed in Remington's Pharmaceutical Sciences, Chapter 84, page 1565,Eighteenth Edition, A. R. Gennaro, (Mack Publishing Co., Easton, Pa.,1990). A pharmaceutical composition comprising ceftolozane can beprepared by adding a stabilizing amount of sodium chloride in a fixedratio to ceftolozane in an aqueous solution prior to lyophilization,then lyophilizing the solution to obtain a lyophilized compositioncomprising sodium chloride and ceftolozane.

In particular, the pharmaceutical antibiotic compositions can includestabilized ceftolozane sulfate obtained by a process comprising thesteps of lyophilizing an aqueous solution containing ceftolozane and astabilizing-effective amount of a stabilizing agent, where thestabilizing-effective amount of the stabilizing agent is about 100 to500 mg (preferably 300-500 mg) of the stabilizing agent per 1,000 mgceftolozane active in the aqueous solution prior to lyophilization. Atherapeutically effective amount of ceftolozane (e.g., ceftolozanesulfate) and a stabilizing-effective amount of the stabilizing agent candissolved in an aqueous solution that can be lyophilized to obtain astabilized ceftolozane pharmaceutical composition.

The method can further comprise the steps of: (1) forming a solutioncomprising sodium chloride and ceftolozane or a salt thereof followed bylyophilizing the solution; and (2) combining the lyophilized ceftolozanewith other components (e.g., a β-lactamase inhibitor, such astazobactam, or a lyophilized β-lactamase inhibitor, such as alyophilized tazobactam) to obtain the pharmaceutical composition. Theresulting pharmaceutical composition can be a powder for reconstitutionto obtain an injectable pharmaceutical composition that can beintravenously administered to a patient. In yet a further embodiment,the method comprises adding 189 mg sodium from sodium chloride per 1000mg of ceftolozane active in an aqueous solution, then lyophilizing thesolution to obtain a lyophilized material comprising sodium chloride andceftolozane sulfate in a ratio effective to provide a product with lessthan 0.03% of the RT63 Impurity as detected by the HPLC method ofExample 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

A. Blending

In other embodiments, pharmaceutical compositions comprising ceftolozanecan be obtained by methods that include the steps of: (1) adding astabilizing amount of sodium chloride to ceftolozane optionally followedby co-lyophilizing or spray drying the ceftolozane and sodium chloride;and (2) combining the product of step (1) with other components. Forexample, the product of step (1) can be combined with a β-lactamaseinhibitor, such as tazobactam (CAS#: 89786-04-9), avibactam(CAS#1192500-31-4), Sulbactam (CAS#68373-14-8) and/or clavulanate(CAS#58001-44-8). The beta lactamase inhibitor can be included in acrystalline or amorpous form, such as a lyophilized tazobactam orcrystalline tazobactam (e.g., U.S. Pat. Nos. 8,476,425 and 5,763,603) toobtain the pharmaceutical composition.

Pharmaceutical compositions comprising ceftolozane and tazobactam withreduced or even undectable levels of the compound of RRT 1.22 (e.g.,including levels of RRT 1.22 that are not detectable by HPLC accordingto Example 1 and/or comprise less than 0.15%, 0.10%, 0.05% or 0.03% byweight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC according toExample 1) can be obtained by blending a first composition comprising atherapeutically effective amount of ceftolozane in the absence oftazobactam with a second composition comprising a therapeuticallyeffective amount of tazobactam in the absence of ceftolozane to form ablended pharmaceutical composition.

Without being bound by theory, the compound RRT 1.22 can be formed by areaction between ceftolozane and formylacetic acid, a by-product oftazobactam as illustrated in Marunaka et al. (Chem. Pharm. Bull. 1988,Vol. 36 (11), pp. 4478-4487.

FIG. 1 is a flowchart showing the steps for preparing a CXA-201composition comprising ceftolozane (referred to as CXA-101) andtazobactam using a blending process, wherein the ceftolozane andtazobactam are lyophilized separately prior to blending as describedherein.

The (first) ceftolozane composition can be prepared in the absence oftazobactam by forming a first aqueous solution comprising ceftolozanesulfate and other components including excipients, stabilizers, pHadjusting additives (e.g., buffers) and the like. Non-limiting examplesof these additives include sodium chloride, citric acid and L-arginine.For example, the use of sodium chloride results in greater stability;L-arginine is used to adjust pH of the aqueous solution (e.g., to pH6-7) and to increase the solubility of ceftolozane; and citric acid isused to prevent discoloration of the product, due to its ability tochelate metal ions. Preferably, the first aqueous solution comprisesabout 125 mg-500 mg sodium chloride per 1,000 mg of ceftolozane active.The ceftolozane can be included as an amount of ceftolozane sulfate offormula (I) containing at least about 1,000 mg ceftolozane active. The(first) aqueous solution is then lyophilized to form a first lyophilizedceftolozane composition, which is combined with tazobactam, e.g., thelyophilized tazobactam (e.g., lyophilized tazobactam sodium) orcrystalline tazobactam.

The (second) tazobactam composition can be prepared in the absence ofceftolozane by forming a second solution comprising tazobactam. Thetazobactam can be included in an amount providing about 500 mg oftazobactam active per 1,000 mg ceftolozane active (i.e., a 1:2 weightratio of tazobactam active to ceftolozane active). Unless otherwiseindicated, tazobactam can be a free acid, a sodium salt, an argininesalt, or a hydrate or solvate thereof. In one embodiment, the tazobactamin the (second) tazobactam composition is tazobactam acid and the secondcomposition further comprises sodium bicarbonate or sodium hydroxide.Lyophilizing tazobactam in the presence of sodium bicarbonate or sodiumhydroxide forms a lyophilized tazobactam sodium, which can then befurther blended with the (first) lyophilized ceftolozane composition.

Pharmaceutical compositions with reduced or undectable amounts of thecompound of RRT 1.22 can be obtained by lyophilizing ceftolozane withoutformylacetic acid and/or tazobactam under conditions that preventformation of RRT 1.22 (e.g., Example 9). The presence of RRT 1.22 can bedetected by HPLC (e.g., Examples 1 and 7). Specific methods oflyophilization are described in Remington's Pharmaceutical Sciences,Chapter 84, page 1565, Eighteenth Edition, A. R. Gennaro, (MackPublishing Co., Easton, Pa., 1990). The formation of the compound offormula (III) can be avoided by preventing the reaction of ceftolozaneand formylacetic acid. In one embodiment, the compound of formula (III)can be suppressed by separately lyophlizing ceftolozane sulfate andtazobactam in separate solutions, and then blending the lyophilizedcompositions to form a pharmaceutical composition.

In one aspect, antibiotic pharmaceutical compositions comprisingceftolozane and tazobactam with less than about 0.15%, 0.10%, 0.05% or0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC ofthe compound of formula (III) are obtained by a process comprising thesteps of: (a) lyophilizing ceftolozane in the absence of tazobactam toobtain a lyophilized ceftolozane composition, and (b) blending thelyophilized ceftolozane composition with a composition comprisingtazobactam under conditions suitable for attaining the aforementionedpurity levels, e.g., by blending with crystalline tazobactam orlyophilized tazobactam.

In another aspect, antibiotic pharmaceutical compositions comprisingceftolozane and tazobactam and less than about 0.15%, 0.10%, 0.05% or0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC ofthe compound of formula (III) are obtained by a process comprising thesteps of: (a) lyophilizing tazobactam in the absence of ceftolozane toobtain a lyophilized tazobactam composition, and (b) blending thelyophilized tazobactam composition with a composition comprisingceftolozane (e.g., lyophilized ceftolozane sulfate).

In a third aspect, antibiotic pharmaceutical compositions comprisingceftolozane and tazobactam and less than about 0.15%, 0.10%, 0.05% or0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC ofthe compound of formula (III) are obtained by a process comprising thesteps of: (a) lyophilizing tazobactam in the absence of ceftolozane toobtain a lyophilized tazobactam composition, (b) lyophilizingceftolozane in the absence of tazobactam to obtain a lyophilizedceftolozane composition, and (c) blending the lyophilized tazobactamcomposition with the lyophilized ceftolozane composition.

Pharmaceutical compositions comprising the compound of formula (III),ceftolozane and tazobactam can be formulated to treat infections byparenteral administration (including subcutaneous, intramuscular, andintravenous) administration. Pharmaceutical compositions mayadditionally comprise excipients, stabilizers, pH adjusting additives(e.g., buffers) and the like. Non-limiting examples of these additivesinclude sodium chloride, citric acid and L-arginine. For example, theuse of sodium chloride results in greater stability; L-arginine is usedto adjust pH and to increase the solubility of ceftolozane; and citricacid is used to prevent discoloration of the product, due to its abilityto chelate metal ions. In one particular embodiment, the pharmaceuticalcompositions described herein are formulated for administration byintravenous injection or infusion.

Other pharmaceutical antibiotic compositions can include ceftolozanesulfate and the compound of formula (III). For example, pharmaceuticalcompositions comprising 0.13%, 0.15%, 0.30%, 0.38%, 0.74% or 0.97% ofthe compound of formula (III) are herein. The pharmaceutical antibioticcompositions can be provided in a unit dosage form (e.g., in acontainer). The unit dosage form can be dissolved with apharmaceutically acceptable carrier, and then intravenouslyadministered. The unit dosage form comprises 1000 mg of ceftolozaneactive and 500 mg tazobactam, typically 1000 mg ceftolozane active asceftolozane sulfate and 500 mg of tazobactam active as tazobactamsodium, argininate or free acid. The unit dosage forms are commonlystored in containers.

In another aspect, provided herein is a unit dosage form of apharmaceutical composition comprising 1,000 mg ceftolozane and 500 mgtazobactam, the pharmaceutical composition formulated for parenteraladministration for the treatment of complicated intra-abdominalinfections or complicated urinary tract infections, the pharmaceuticalcomposition comprising ceftolozane sulfate and tazobactam, obtained by aprocess comprising the steps of: lyophlizing an aqueous solution toobtain a lyophilized ceftolozane composition, wherein the aqueoussolution comprises water, ceftolozane sulfate, 125-500 mg sodiumchloride per 1,000 mg ceftolozane active in the aqueous solution, anamount of L-arginine to provide a pH of 6-7 in the solution prior tolyophilization; and blending the lyophilized ceftolozane compositionwith a lyophilized tazobactam composition in an amount providing theratio of about 500 mg tazobactam free acid per 1,000 mg of ceftolozaneactive to obtain the unit dosage form.

Another embodiment of the invention is a container containing a unitdosage form of a pharmaceutical composition formulated for parenteraladministration for the treatment of complicated intra-abdominalinfections or complicated urinary tract infections. The container can beobtained by a process comprising the steps of: a) lyophilizing anaqueous solution comprising 189 mg sodium from sodium chloride per 1000mg ceftolozane active in the form of ceftolozane sulfate and furthercomprising citric acid, and L-arginine to obtain a lyophilizedceftolozane composition; and b) filling a sufficient quantity of thelyophilized composition into a container to obtain a unit dosage formcomprising 189 mg sodium from sodium chloride and 1,000 mg ofceftolozane active in the form of ceftolozane sulfate. In one aspect,the pH of the aqueous solution is 6.0 to 7.0. In another aspect thepharmaceutical composition is formulated for parenteral administrationby reconstituting the pharmaceutical composition in the container (e.g.,with 10 mL of diluent such as water for injection or isotonic saline)followed by addition of the reconstituted pharmaceutical composition toa carrier for injection (e.g., about 100 mL of isotonic saline or otherpharmaceutically acceptable carrier for intravenous administration).Optionally, the container is also filled with tazobactam (e.g., alyophilized tazobactam such as tazobactam sodium). In yet anotheraspect, the pharmaceutical composition is a liquid compositioncomprising 189 mg sodium from sodium chloride, 1,000 mg of ceftolozaneactive and tazobactam in an amount providing about 500 mg tazobactamacid equivalent per 1,000 mg of ceftolozane active, formulated forparenteral administration and the pH of the aqueous solution is 6.0 to7.0.

The pharmaceutical composition in the container can also be aCeftolozane/Tazobactam for Injection Drug Product, 1000 mg/500 mg. It ispresented as a combination of two sterile active powders in a singlecontainer intended for reconstitution and intravenous infusion. In anembodiment, the drug product is prepared by converting ceftolozanesulfate to a sterile drug product intermediate (composition) powder withexcipients citric acid, sodium chloride and L-arginine. This can be doneby lyophilization, as described herein. Tazobactam sodium drug substancecan be presented as a sterile powder without any excipients. Thetazobactam sodium drug substance can be lyophilized, spray dried orprovided as a crystalline material. The drug product is then prepared byaseptically filling the two powders (e.g., the two separatelylyophilized drug powders) sequentially into a single container.

In an embodiment, the container of ceftolozane/tazobactam for injectioncontains approximately 2255 mg ceftolozane sterile composition powderthat contains 1147 mg ceftolozane sulfate, which is equivalent to 1000mg ceftolozane free base, as well as approximately 537 mg tazobactamsodium sterile drug substance, equivalent to 500 mg tazobactam freeacid. At the time of administration, the container is reconstituted with10 mL vehicle, sterile 5% Dextrose Injection USP, Water for Injection or0.9% Sodium Chloride Injection USP, then the container contents furtherdiluted in an infusion bag of 0.9% Sodium Chloride Injection USP or 5%Dextrose Injection USP, for administration. The constituents are shownin Table 29.

A pharmaceutical composition can include ceftolozane sulfate andtazobactam in an amount providing 1,000 mg of ceftolozane active per 500mg of tazobactam active, and 0.03% to 0.15% by HPLC of a compound offormula (III) detectable at a retention time relative to ceftolozane of1.22 by high performance liquid chromatography using the methoddescribed in Example 1. Optionally, the pharmaceutical composition canfurther include 125 mg to 500 mg of sodium chloride per 1,000 mg ofceftolozane active, and L-arginine. The tazobactam in the compositioncan be tazobactam sodium.

In one embodiment of these methods of preparing, 125-500 mg sodiumchloride per 1000 mg of ceftolozane is combined. In another embodimentof these methods of preparing, the amount of the sodium chloridecombined is 200-500 mg sodium chloride per 1000 mg of ceftolozane,300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodiumchloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg ofceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane.In another embodiment of these methods of preparing, the amount of thesodium chloride combined is about 487 mg sodium chloride per 1000 mg ofceftolozane.

In another embodiment of these methods of preparing, the method furthercomprises lyophilizing the ceftolozane in the absence of the tazobactam.In yet another embodiment, the method can further comprise lyophilizingthe tazobactam in the absence of the ceftolozane.

Accordingly, in one aspect, provided herein is a pharmaceuticalcomposition comprising ceftolozane and tazobactam, wherein thecomposition comprises less than 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.15%,0.1%, or 0.05% by weight of the compound RRT 1.22. In another aspect,provided herein is a pharmaceutical composition comprising ceftolozaneand tazobactam, wherein the composition comprises less than 0.1% byweight of the compound RRT 1.22. In one embodiment, the pharmaceuticalcomposition comprises less than 0.05% by weight of the compound RRT1.22. In another embodiment, the pharmaceutical composition comprisesless than 0.15% by weight of the compound RRT 1.22. In yet anotherembodiment, the pharmaceutical composition comprises no detectableamount of the compound RRT 1.22 as measured by HPLC.

In contrast, a greater amount of compound RRT 1.22 was found incompositions of ceftolozane and tazobactam, wherein the compositionswere formed through co-lyophilization, i.e., the ceftolozane andtazobactam were combined and co-lyophilized together (see, e.g.,Examples 6, 7a, and 7b), as opposed to being individually lyophilizedand blended together (see, e.g., Examples 9 and 10).

In one aspect, provided herein is an antibacterial pharmaceuticalcomposition comprising ceftolozane sulfate and tazobactam in a ratio of1,000 mg ceftolozane active per 500 mg of tazobactam active, thepharmaceutical composition obtained by a process comprising the stepsof: lyophilizing a first aqueous solution in the absence of tazobactam,the first aqueous solution comprising ceftolozane sulfate prior tolyophilization to obtain a first lyophilized ceftolozane composition;and blending the first lyophilized ceftolozane composition withtazobactam to obtain an antibacterial composition comprising less than0.13% by HPLC of a compound of formula (III) (compound RRT 1.22)detectable at a retention time relative to ceftolozane of 1.22 by highperformance liquid chromatography using the method described in Example1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25

In one embodiment, the antibacterial composition comprises less than0.03% of the compound of formula (III) detected by HPLC. In anotherembodiment, the first aqueous solution further comprises L-arginine inan amount effective to adjust the pH of the first aqueous solution to6-7 prior to lyophilization to obtain a first lyophilized ceftolozanecomposition.

In another embodiment, the antibacterial pharmaceutical composition isobtained by a process further comprising the steps of: lyophilizing asecond solution comprising tazobactam in the absence of ceftolozane toform a second lyophilized tazobactam composition; and blending the firstlyophilized ceftolozane composition and the second lyophilizedtazobactam composition to obtain the antibacterial composition.

In another embodiment, the tazobactam in the second solution istazobactam acid, and wherein the tazobactam acid in the second solutionis lyophilized in the presence of sodium bicarbonate to form the secondlyophilized tazobactam solution.

In another embodiment, the first aqueous solution comprises L-argininein an amount effective to provide a pH of about 5-7, e.g., 6-7. Inanother embodiment, the first aqueous solution comprises 125 mg to 500mg of sodium chloride per 1,000 mg of ceftolozane active.

In another embodiment, the first aqueous solution further comprisescitric acid. In another embodiment, the first aqueous solution consistsof ceftolozane sulfate, citric acid, sodium chloride, L-arginine, andwater.

In another aspect, provided herein is a unit dosage form of apharmaceutical composition formulated for parenteral administration forthe treatment of complicated intra-abdominal infections or complicatedurinary tract infections, the pharmaceutical composition comprisingceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozaneactive per 500 mg of tazobactam active, the pharmaceutical compositionobtained by a process comprising the steps of: lyophilizing a firstaqueous solution in the absence of tazobactam, the first aqueoussolution comprising ceftolozane sulfate, 125 mg to 500 mg of sodiumchloride per 1,000 mg of ceftolozane active, at a pH of 6-7 prior tolyophilization to obtain a first lyophilized ceftolozane composition;lyophilizing a second solution comprising tazobactam in the absence ofceftolozane to form a second lyophilized tazobactam composition; andblending the first lyophilized ceftolozane composition and the secondlyophilized tazobactam composition to obtain the antibacterialcomposition.

In another embodiment, the unit dosage form comprises a total of notmore than 0.03% by HPLC of a compound of formula (III) detectable at aretention time relative to ceftolozane of 1.22 by high performanceliquid chromatography using the method described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25to detect:

In another embodiment, the unit dosage form comprises a total of 1,000mg of ceftolozane active and a total of 500 mg of tazobactam active.

In another embodiment, the unit dosage form comprises a total of notmore than 0.03% by HPLC of a compound of formula (III) detectable at aretention time relative to ceftolozane of 1.22 by high performanceliquid chromatography using the method described in Example 1, i.e.:

Column Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm Column temperature 45° C.± 2° C. Wavelength: 254 nm for ceftolozane and ceftolozane-relatedsubstances in Table 1A Mobile phase A 50 mM Sodium PerchlorateMonohydrate in water, adjusted to pH 2.5 with Diluted Perchloric AcidMobile phase B 63 mM Sodium Perchlorate Monohydrate in water, adjustedto pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v) Time(min.) % A % B Gradient Profile 0 75 25 30 70 30 60 0 100 85 0 100 85.175 25 110 75 25to detect:

In another aspect, provided herein is a compound of formula (III):

or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is a pharmaceutical compositioncomprising a compound of formula (III):

or a pharmaceutically acceptable salt thereof.

In one embodiment, the pharmaceutical composition further comprisesceftolozane sulfate. In another embodiment, the pharmaceuticalcomposition further comprises tazobactam.

In another embodiment, the compound of formula (III) is obtained by aprocess comprising the step of reacting ceftolozane and formylaceticacid to obtain the compound of formula (III). In another embodiment, thecompound of formula (III) is obtained by a process comprising the stepof reacting ceftolozane and tazobactam acid to obtain the compound offormula (III).

In another embodiment, the compound of formula (III) is obtained by aprocess comprising the steps of: forming an aqueous solution comprisingceftolozane and tazobactam acid; and lyophilizing the aqueous solutionto obtain a lyophilized composition comprising the compound of formula(III). In another embodiment, the aqueous solution comprises ceftolozanesulfate and tazobactam acid in a 2:1 weight ratio between the amount ofceftolozane active and the amount of tazobactam acid. In anotherembodiment, the aqueous solution comprises sodium chloride, ceftolozanesulfate, tazobactam acid and L-arginine.

In another embodiment, the aqueous solution has a pH of about 5.0 to7.0, e.g., 6.0 to 7.0. In another embodiment, the pharmaceuticalcomposition is formulated for parenteral administration. In anotherembodiment, the compound of formula (III) is obtained by a processfurther comprising the step of performing high performance liquidchromatography (HPLC) on the lyophilized composition to isolate thecompound of formula (III).

In another embodiment, the pharmaceutical composition comprises about0.13-0.97% of the compound of formula (III). In another embodiment, thepharmaceutical composition comprises up to about 0.15% of the compoundof formula (III). In another aspect, provided herein is a pharmaceuticalcomposition comprising a compound of formula (III),

the compound of formula (III) obtained by a process comprising the stepsof: forming an aqueous solution comprising tazobactam acid andceftolozane sulfate in an amount providing 1,000 mg of ceftolozaneactive per 500 mg of tazobactam acid in the aqueous solution;lyophilizing the aqueous solution of step (a) to obtain a lyophilizedcomposition comprising a compound of formula (III); and formulating thelyophilized composition as a pharmaceutical composition for parenteraldelivery.

In one embodiment, the pH of the aqueous solution is 5.0 to 7.0, e.g.,6.0 to 7.0. In another embodiment, the pharmaceutical composition isformulated for parenteral administration.

In another aspect, provided herein is a pharmaceutical compositionformulated for parenteral administration for the treatment ofcomplicated intra-abdominal infections or complicated urinary tractinfections, the pharmaceutical composition comprising a compound offormula (III) in a lyophilized composition obtained by lyophilizing anaqueous solution comprising tazobactam and an amount of ceftolozanesulfate containing 1,000 mg of ceftolozane active per 500 mg oftazobactam acid.

B. Co-Lyophilization

In one aspect, provided herein is a method of preparing a compositioncomprising ceftolozane and sodium chloride, comprising combining sodiumchloride with ceftolozane, wherein 125-1000 mg sodium chloride per 1000mg of ceftolozane is combined, followed by lyophilization of the sodiumchloride ceftolozane mixture. The process is referred to herein as“co-lyophilization”. In another aspect, provided herein is a method ofpreparing a composition comprising sodium chloride, tazobactam, andceftolozane, comprising combining sodium chloride, tazobactam, andceftolozane, wherein 125-1000 mg sodium chloride per 1000 mg ofceftolozane is combined, followed by lyophilization of the mixture ofsodium chloride, tazobactam, and ceftolozane.

Also provided herein is a method of preparing a pharmaceuticalcomposition comprising sodium chloride, ceftolozane, and tazobactam,comprising combining sodium chloride, tazobactam, and ceftolozane,followed by spray-drying the mixture of sodium chloride, ceftolozane,and tazobactam.

FIG. 2 is a flowchart showing the steps for preparing a CXA-201composition comprising ceftolozane (referred to as CXA-101) andtazobactam using a co-lyophilization process, as described herein.

In another aspect, provided herein is a pharmaceutical compositioncomprising stabilized ceftolozane sulfate obtained by a processcomprising lyophilizing an aqueous solution comprising 125 mg to 500 mgsodium chloride with an amount of ceftolozane sulfate providing 1,000 mgof ceftolozane active, to obtain the lyophilized stabilized ceftolozanesulfate composition.

In one embodiment, the stabilized ceftolozane is obtained bylyophilizing the sodium chloride and ceftolozane sulfate withL-arginine. In another embodiment, the stabilized ceftolozane isobtained by lyophilizing an aqueous solution having a pH of about 5.0 to7.0, e.g., 6.0 to 7.0.

In another embodiment, the stabilized ceftolozane is obtained bylyophilizing the sodium chloride and ceftolozane sulfate with L-arginineand citric acid. In another embodiment, the pharmaceutical compositionis formulated for parenteral administration. In another embodiment, thecomposition is a unit dosage form in a container comprising 125 mg to500 mg sodium chloride, 1,000 mg of ceftolozane in the form ofceftolozane sulfate, and L-arginine. In another embodiment, thepharmaceutical composition is formulated for parenteral administration.In another embodiment, the pH of the aqueous solution is 6.0 to 7.0.

In another aspect, provided herein is a container comprising apharmaceutical composition of stabilized ceftolozane sulfate, obtainedby a process comprising the step of: lyophilizing an aqueous solutioncomprising 125 mg to 500 mg sodium chloride with an amount ofceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtainthe lyophilized stabilized ceftolozane sulfate composition; filling thelyophilized stabilized ceftolozane composition into a container.

IV. Manufacturing for the Prevention of Cross-Contamination

Recent FDA manufacturing guidance (published in April 2013) states thatmanufacturing facilities dedicated to manufacturing a sensitizingnon-penicillin beta-lactam compound should be “completely andcomprehensively separated” from areas in the facility in which any classof sensitizing beta-lactam is manufactured. See U.S. Department ofHealth and Human Services Food and Drug Administration, Center for DrugEvaluation and Research, Non-Penicillin Beta-Lactam Drugs: A CGMPFramework for Preventing Cross-Contamination (April 2013) (“FDAGuidance”). The FDA also considers separation of production facilitiesfor penicillins to be good manufacturing practice. The FDA Guidance canbe understood to require the use of a dedicated facility to manufactureantibiotic compounds comprising a non-penicillin beta-lactam compound(e.g., a cephalosporin) and a BLI compound with a beta-lactam ring(e.g., tazobactam). Accordingly, a facility that manufactures a productcontaining both cephalosporin and a beta-lactam containing BLI such astazobactam for sale in the United States cannot be subsequently used tomanufacture any other products containing beta-lactam ring, other thanadditional combinations of other cephalosporins with the same BLIcompound (e.g., other non-penicillin beta-lactam compounds includingother cephalosporin antibitoics cannot be subsequently manufactured inthe facility).

Beta-lactam antibiotics, including penicillin and the non-penicillinclasses, share a basic chemical structure that includes a three-carbon,one-nitrogen cyclic amine structure known as the beta-lactam ring. Theside chain associated with the beta-lactam ring is a variable groupattached to the core structure by a peptide bond; the side chainvariability contributes to antibacterial activity. As of the date ofthis publication, FDA has approved over 34 beta-lactam compounds asactive ingredients in drugs for human use. (see, e.g., FDA's ApprovedDrug Products with Therapeutic Equivalence Evaluations, generally knownas the Orange Book) Beta-lactam antibiotics include the following fiveclasses: penicillins (e.g., ampicillin, oxacillin); cephalosporins(e.g., cephalexin, cefaclor); penems (e.g., imipenem, meropenem);carbacephems (e.g., loracarbef); and monobactams (e.g., aztreonam).(Yao, JDC, and RC Moellering, Jr., Antibacterial agents, in Manual ofClinical Microbiology, 9th edition, edited by PR Murray et al.,Washington D.C., ASM Press, 2007.)

Under the FDA Guidance, a manufacturing facility handling a product forsale in the United States containing both a cephalosporin (e.g,ceftolozane) and a penicillin nucleus (e.g., tazobactam) cannot besubsequently used in the manufacture of any other class of beta-lactamproducts, including all other penicillins, cephalosporins, penems,carbacephems and monobactams or in the manufacture of other finishedpharmaceuticals or active pharmaceutical ingredients. The FDA Guidancestates that (non-penicillin) cephalosporin beta-lactam compounds (e.g.,such as ceftolozane) for sale in the United States must be “completelyand comprehensively separated from” manufacturing areas that handle anyother class of beta-lactam compound (e.g., compounds in the penicillinclass).

A product containing ceftolozane and tazobactam includes both anon-penicillin beta-lactam cephalosporin (ceftolozane) and abeta-lactamase inhibitor with a beta-lactam moiety (tazobactam). Underthe FDA Guidance, these two compounds must be “completely andcomprehensively separated.” Accordingly, there is a need for methods ofmanufacturing antibiotic compositions comprising ceftolozane andtazobactam for sale in the United States in compliance with the FDAGuidance, as well as antibiotic compositions manufactured in accordancewith the FDA Guidance without affecting the purity, stability, andsafety of the resulting composition.

Provided herein are methods of manufacturing or preparing pharmaceuticalcompositions containing two or more beta-lactam compounds in accordancewith FDA Guidance, as well as pharmaceutical compositions manufacturedin compliance with FDA Guidance. Specifically, certain manufacturingmethods are provided herein that conform to standards recommended by FDAGuidance for the avoidance of cross-contamination of non-penicillinbeta-lactam drugs.

In one aspect, provided herein is an antibacterial pharmaceuticalcomposition formulated for parenteral administration for the treatmentof infections, the pharmaceutical composition comprising atherapeutically effective amount of ceftolozane sulfate and tazobactamin a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactamactive, the pharmaceutical composition obtained by a process comprisingthe steps of:

-   -   a. lyophilizing a first aqueous solution in the absence of        tazobactam, the first aqueous solution comprising ceftolozane        sulfate, to obtain a first lyophilized ceftolozane composition;    -   b. blending the lyophilized ceftolozane composition with a        tazobactam composition comprising tazobactam prepared and        provided in the absence of ceftolozane;

wherein the process is completed in the absence of othernon-cephalosporin beta-lactam compounds.

In another aspect, provided herein is a unit dosage form of apharmaceutical composition formulated for parenteral administration forthe treatment of complicated intra-abdominal infections or complicatedurinary tract infections, the pharmaceutical composition comprisingceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozaneactive per 500 mg of tazobactam active, the pharmaceutical compositionobtained by a process comprising the steps of

-   -   a. lyophilizing a first aqueous solution in the absence of        tazobactam, the first aqueous solution comprising ceftolozane        sulfate, 125 mg to 500 mg of sodium chloride per 1,000 mg of        ceftolozane active, to obtain a first lyophilized ceftolozane        composition,    -   b. lyophilizing a second solution comprising tazobactam in the        absence of ceftolozane to form a second lyophilized tazobactam        composition; and    -   c. blending the first lyophilized ceftolozane composition and        the second lyophilized tazobactam composition to obtain the        antibacterial composition;

wherein the process is completed in the absence of othernon-cephalosporin beta-lactam compounds.

V. Methods of Treatment

Pharmaceutical compositions comprising ceftolozane/tazobactam are beingdeveloped as an intravenous (IV) formulation for the treatment ofcomplicated urinary tract infections (cUTIs) and complicatedintra-abdominal infections (cIAIs).

Ceftolozane/tazobactam is an antibacterial composition includingceftolozane, a cephalosporin with potent antipseudomonalcephalosporinactivity, in combination with tazobactam, a beta(β)-lactamase inhibitor (BLI). Like other members of the cephalosporinclass, ceftolozane is believed to exert its bactericidal activity byinhibiting essential penicillin-binding proteins (PBPs), resulting ininhibition of cell wall synthesis and subsequent cell death. Ceftolozanehas activity against Pseudomonas aeruginosa including strains that areresistant to carbapenems, cephalosporins, fluoroquinolones, andaminoglycosides, and other common Gram-negative pathogens, includingmost extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae.Tazobactam inhibits chromosomal- and plasmid-mediated bacterial class Aand C β lactamases. Tazobactam is believed to protect ceftolozane fromhydrolysis by covalently binding these enzymes, and broadens coverage toinclude most ESBL-producing Escherichia coli, Klebsiella pneumoniae, andother Enterobacteriaceae, including some Enterobacteriaceaeoverexpressing AmpC. Tazobactam inhibits or decreases the activity ofbeta-lactamases (e.g., bacterial beta-lactamases), and can be combinedwith beta-lactam compounds (e.g., antibiotics), thereby broadening thespectrum of the beta-lactam compound and increasing the beta-lactamcompound's efficacy against organisms that produce beta-lactamase. Acompound or a composition possesses efficacy against an organism if itkills or weakens the organism, or inhibits or prevents reproduction theorganism.

The CXA-201 product (ceftolozane/tazobactam for injection) is undergoingregulatory review for the treatment of complicated intra-abdominalinfections (cIAI) caused by susceptible isolates of the Gram-negativeand Gram-positive microorganisms such as: Citerobacter freundii,Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia, Klebsiellaoxytoca, Proteus mirabilis, and Pseudomonas aeruginosa. In patients whoare at risk of mixed aerobic-anaerobic infection, concurrent therapywith an anti-anaerobic agent can also be used.

The CXA-201 product (ceftolozane/tazobactam for injection) is undergoingregulatory review for the treatment of complicated urinary tractinfections (cUTI), including pyelonephritis caused by susceptibleisolates of the following Gram-negative microorganisms: Enterobacterspp, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis andPseudomonas aeruginosa. In one embodiment, a pharmaceutical compositioncomprising a CXA-201 product (e.g, the unit dosage container of Table 29below) is reconstituted in a pharmaceutically acceptable carrier (e.g.,a total volume of about 90-150 mL, preferably about 110 mL, of 0.9%aqueous sodium chloride for injection or in initial volume of 10-20 mLof water for injection or 0.9% aqueous sodium chloride for injection,followed by dilution of this solution into a 100 mL volume of 0.9%aqueous sodium chloride for injection). The resulting pharmaceuticalcomposition can be infused into a patient in need thereof for treatmentof a complicated intra-abdominal infection (e.g, using 1 hour infusiontimes) three times per day (e.g., once every 8 hours) for a recommendedduration of treatment (e.g. 4-10 days).

The preferred dosage of (ceftolozane/tazobactam for injection) for cUTIand cIAI is 1.5 g administered every 8 hours by intravenous (IV)infusion over 1 hour in patients ≥18 years of age. The duration oftherapy should be guided by the severity and site of infection and thepatient's clinical and bacteriological progress. In one embodiment, apharmaceutical composition comprising a CXA-201 product (e.g, the unitdosage container of Table 29 below) is reconstituted in apharmaceutically acceptable carrier (e.g., a total volume of about90-150 mL, preferably about 110 mL, of 0.9% aqueous sodium chloride forinjection or in initial volume of 10-20 mL of water for injection or0.9% aqueous sodium chloride for injection, followed by dilution of thissolution into a 100 mL volume of 0.9% aqueous sodium chloride forinjection). The resulting pharmaceutical composition can be infused intoa patient in need thereof for treatment of a Complicated Urinary TractInfections (cUTI), Including Pyelonephritis (e.g, using 1 hour infusiontimes) three times per day (e.g., once every 8 hours) for a recommendedduration of treatment (e.g. 7 days).

Ceftolozane/tazobactam displays potent antibacterial activity againstcommon Gram-negative organisms, including Enterobacteriaceae andPseudomonas aeruginosa; select Gram-positive organisms, includingstreptococci; the majority of pathogenic enteric bacilli and selectGram-positive anaerobic species, thus making ceftolozane/tazobactam apotentially practical choice for pathogens involved ingastro-intestinal, urinary and community acquired as well as nosocomialrespiratory infections. In general, the Gram-positive and Gram-negativespectrum of ceftolozane is similar to ceftazidime, but itsantipseudomonal activity is the most potent among all currentlyavailable β-lactams, including the cephalosporins and carbapenems. Mostimportantly, ceftolozane has been shown to be active against strains ofP. aeruginosa that are resistant to carbapenems, cephalosporins,fluoroquinolones, and aminoglycosides, including the majority ofmulti-drug resistant isolates. Indeed, the minimum inhibitoryconcentration (MIC) required to inhibit the growth of 90% of organisms(MIC90) for P. aeruginosa (MIC90≤2 μg/mL) is the lowest among allsystemically administered antipseudomonal antibiotics.

In vitro studies have demonstrated that ceftolozane/tazobactam has abroad spectrum of activity against Gram-negative bacteria. The in vitroactivity of ceftolozane and ceftolozane/tazobactam was evaluated againsta broad range of Gram-positive and Gram-negative bacteria. It wasobserved that tazobactam potentiated the activity of ceftolozane againstAcinetobacter spp. and common species of Enterobacteriaceae, includingCitrobacter spp., Enterobacter cloacae, E. coli, K. pneumoniae, Proteusmirabilis, and Serratia marcescens. These surveillance data demonstratethat 88% to 100% of these Enterobacteriaceae species are inhibited at <8μg/mL.

In one aspect, provided herein is a method for the treatment ofbacterial infections in a mammal, comprising administering to saidmammal a therapeutically effective amount of a pharmaceuticalcomposition prepared according to the methods described herein. Inanother aspect, provided herein is a method for the treatment ofbacterial infections in a mammal, comprising administering to saidmammal a therapeutically effective amount of tazobactam and ceftolozane.In certain embodiments of the above methods, the bacterial infection iscaused by an extended-spectrum beta-lactamase-producing organism. Incertain embodiments, the bacterial infection is caused by anantibiotic-resistant organism. In yet another aspect, the invention is amethod for the treatment of bacterial infections in a mammal, comprisingadministering to said mammal a therapeutically effective amount of apharmaceutical composition comprising both tazobactam and ceftolozane.In certain embodiments of the above methods, the bacterial infection iscaused by an extended-spectrum beta-lactamase-producing organism. Incertain embodiments, the bacterial infection is caused by anantibiotic-resistant organism.

In another aspect, provided herein is a method for the treatment ofbacterial infections in a mammal, comprising administering to saidmammal a therapeutically effective amount of a pharmaceuticalcomposition comprising tazobactam, ceftolozane, and less than 0.1% byweight of the compound RRT 1.22. In another embodiment of the treatmentmethod, the pharmaceutical composition comprises tazobactam,ceftolozane, and less than 0.05% by weight of the compound RRT 1.22.

In certain embodiments of the treatment methods, the pharmaceuticalcomposition further comprises 125 to 1000 mg sodium chloride per 1000 mgof ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg ofceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane,300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodiumchloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg ofceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane.In one specific embodiment of the treatment methods, the pharmaceuticalcomposition further comprises about 487 mg sodium chloride per 1000 mgof ceftolozane.

In other embodiments of the treatment methods, the pharmaceuticalcomposition comprises 250-750 mg tazobactam per 1000 mg of ceftolozane,e.g., 250-700 mg tazobactam per 1000 mg of ceftolozane, 300-700 mgtazobactam per 1000 mg of ceftolozane, 300-650 mg tazobactam per 1000 mgof ceftolozane, 350-650 mg tazobactam per 1000 mg of ceftolozane,350-600 mg tazobactam per 1000 mg of ceftolozane, 400-600 mg tazobactamper 1000 mg of ceftolozane, 400-550 mg tazobactam per 1000 mg ofceftolozane, 450-550 mg tazobactam per 1000 mg of ceftolozane, or about500 mg tazobactam per 1000 mg of ceftolozane.

Non-limiting examples of the bacterial infections that can be treated bythe methods of the invention include infections caused by: aerobic andfacultative gram-positive microorganisms (e.g., Staphylococcus aureus,Enterococcus faecalis, Staphylococcus epidermidis, Streptococcusagalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Viridansgroup streptococci), aerobic and facultative gram-negativemicroorganisms (e.g., Acinetobacter baumanii, Escherichia coli,Haemophilus influenza, Klebsiella pneumonia, Pseudomonas aeruginosa,Citrobacter koseri, Moraxella catarrhalis, Morganella morganii,Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Serratiamarcescens, Providencia stuartii, Providencia rettgeri, Salmonellaenterica), gram-positive anaerobes (Clostridium perfringens), andgram-negative anaerobes (e.g., Bacteroides fragilis group (e.g., B.fragilis, B. ovatus, B. thetaiotaomicron, and B. vulgates), Bacteroidesdistasonis, Prevotella melaninogenica).

In certain embodiments of the methods described herein, the bacterialinfections resulting from beta-lactamase-producing organisms are treatedor controlled. Non-limiting examples of beta-lactamase-producingorganisms include:

(1) ESBL (extended-spectrum beta-lactamase)-producing organisms selectedfrom the group consisting of Enterobacteriaceae spp.: Escherichia coli,Klebsiella spp. (including K. pneumoniae and K. oxytoca), Proteusmirabilis, Proteus vulgaris, Enterobacter spp., Serratia spp.,Citrobacter spp., Pseudomonas spp., Acinetobacter spp.) and Bacteroidesspp.;

(2) CSBL (conventional-spectrum beta-lactamase)-producing organisms,known to those of skill in the art; and

(3) Inducible-AmpC-type beta-lactamases, such as Citrobacter spp.,Serratia spp., Morganella morganii, Proteus vulgaris, and Enterobactercloacae.

In certain embodiments of the methods described herein, the bacterialinfection is associated with one or more of the following conditions:

Appendicitis (complicated by rupture or abscess) and peritonitis causedby piperacillin-resistant beta-lactamase producing strains ofEscherichia coli or the following members of the Bacteroides fragilisgroup: B. fragilis, B. ovatus, B. thetaiotaomicron, or B. vulgates;

Uncomplicated and complicated skin and skin structure infections,including cellulitis, cutaneous abscesses, and ischemic/diabetic footinfections caused by piperacillin-resistant, beta-lactamase producingstrains of Staphylococcus aureus;

Postpartum endometritis or pelvic inflammatory disease caused bypiperacillin-resistant, beta-lactamase producing strains of Escherichiacoli;

Community-acquired pneumonia (moderate severity only) caused bypiperacillin-resistant, beta-lactamase producing strains of Haemophilusinfluenza;

Nosocomial pneumonia (moderate to severe) caused bypiperacillin-resistant, beta-lactamase producing strains ofStaphylococcus aureus and by Acinetobacter baumanii, Haemophilusinfluenzae, Klebsiella pneumoniae, and Pseudomonas aeruginosa.Nosocomial pneumonia is also known as hospitalacquired/ventilator-associated bacterial pneumonia (HABP/VABP);

Complicated intra-abdominal infections (cIAI);

Complicated urinary tract infections (cUTIs);

Acute Pyelonephritis; and

Systemic Inflammatory Response Syndrome (SIRS).

Also provided herein is the use of tazobactam, and hydrates and solvatesthereof, in combination with ceftolozane, for the preparation of amedicament for the treatment of bacterial infections. The bacterialinfections can result from either gram-negative or gram-positiveorganisms.

The compositions provided herein can be used in the treatment ofinfections caused by Pseudomonas aeruginosa, Serratia marcescens,Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, orStreptococcus pneumonia.

In one embodiment of the treatment methods, the bacterial infections areGram-negative bacterial infections. In one embodiment, the gram-negativeinfections are complicated Urinary Tract Infections (cUTI) andcomplicated intra-abdominal infections (cIAI). In another embodiment,the gram-negative bacterial infections are caused by Pseudomonasaeruginosa, E. coli, and/or Klebsiella pneumonia.

In a further embodiment, provided herein is a method for the treatmentof gram-negative bacterial infections in a mammal, comprisingadministering to said mammal a therapeutically effective amount of apharmaceutical composition comprising ceftolozane, about 500 mgtazobactam free acid per 1000 mg of ceftolozane, about 476 mg sodiumchloride per 1000 mg of ceftolozane, about 587 mg L-arginine per 1000 mgof ceftolozane, and about 21 mg anhydrous citric acid per 1000 mg ofceftolozane. In one embodiment, the gram-negative bacterial infectionsare selected from the group consisting of complicated Urinary TractInfections (cUTI) and complicated intra-abdominal infections (cIAI). Inanother embodiment, the gram negative bacterial infection is nosocomialpneumonia.

In another specific embodiment, provided herein is a method for thetreatment of gram-negative bacterial infections in a mammal, comprisingadministering to said mammal a therapeutically effective amount of apharmaceutical composition comprising ceftolozane, about 500 mgtazobactam free acid equivalents per 1000 mg of ceftolozane, about 487mg sodium chloride per 1000 mg of ceftolozane, about 600 mg L-arginineper 1000 mg of ceftolozane, and about 21 mg anhydrous citric acid per1000 mg of ceftolozane. In one embodiment, the gram-negative bacterialinfections are selected from the group consisting of complicated UrinaryTract Infections (cUTI) and complicated intra-abdominal infections(cIAI). In another embodiment, the gram negative bacterial infection isnosocomial pneumonia.

In one embodiment, provided herein is a method for the treatment of aninfection in a mammal, wherein the infection is caused by Pseudomonasaeruginosa, Serratia marcescens, Escherichia coli, Klebsiellapneumoniae, Haemophilus influenzae, or Streptococcus pneumoniaecomprising administering to said mammal a therapeutically effectiveamount of a pharmaceutical composition comprising ceftolozane,corresponding to approximately 1000 mg of the free base form ofceftolozane; tazobactam, corresponding to approximately 500 mg of thetazobactam acid form; and 400-500 mg sodium chloride. In one embodiment,the pharmaceutical composition further comprises 500-650 mg L-arginineand 15-30 mg anhydrous citric acid.

In another embodiment, provided herein is a method for the treatment ofurinary tract infection, intra-abdominal infection, or nosocomialpneumonia in a mammal, comprising administering to said mammal atherapeutically effective amount of a pharmaceutical compositioncomprising ceftolozane, corresponding to approximately 1000 mg of thefree base form of ceftolozane; tazobactam, corresponding toapproximately 500 mg of the tazobactam acid form; and 400-500 mg sodiumchloride. In an embodiment, the pharmaceutical composition comprises 487mg sodium chloride. In one embodiment, the pharmaceutical compositionfurther comprises 500-650 mg L-arginine and 15-30 mg anhydrous citricacid.

In one embodiment, provided herein is a method for the treatment of aninfection in a mammal, wherein the infection is caused by Pseudomonasaeruginosa, Serratia marcescens, Escherichia coli, Klebsiellapneumoniae, Haemophilus influenzae, or Streptococcus pneumoniaecomprising administering to said mammal a therapeutically effectiveamount of a pharmaceutical composition comprising approximately 1147 mgceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500mg sodium chloride. In one embodiment, the pharmaceutical compositionfurther comprises 500-650 mg L-arginine and 15-30 mg anhydrous citricacid.

In another embodiment, provided herein is a method for the treatment ofurinary tract infection, intra-abdominal infection, or nosocomialpneumonia in a mammal, comprising administering to said mammal atherapeutically effective amount of a pharmaceutical compositioncomprising approximately 1147 mg ceftolozane sulfate; approximately 537mg tazobactam sodium; and 400-500 mg sodium chloride. In an embodiment,the pharmaceutical composition comprises 487 mg sodium chloride. In oneembodiment, the pharmaceutical composition further comprises 500-650 mgL-arginine and 15-30 mg anhydrous citric acid.

In one embodiment, provided herein is a method for the treatment of aninfection in a mammal, wherein the infection is caused by Pseudomonasaeruginosa, Serratia marcescens, Escherichia coli, Klebsiellapneumoniae, Haemophilus influenzae, or Streptococcus pneumoniaecomprising administering to said mammal a therapeutically effectiveamount of a pharmaceutical composition comprising approximately 1147 mgceftolozane sulfate; approximately 537 mg tazobactam sodium;approximately 487 mg sodium chloride; and approximately 600 mgL-arginine. In one embodiment, the pharmaceutical composition furthercomprises 15-30 mg anhydrous citric acid.

As used herein, “treating”, “treat” or “treatment” describes themanagement and care of a patient for the purpose of combating a disease,condition, or disorder and includes the administration of apharmaceutical composition of the present invention to alleviate thesymptoms or complications of a disease, condition or disorder, or toeliminate the disease, condition or disorder. The term “treat” can alsoinclude treatment of a cell in vitro or an animal model.

By a “therapeutically effective amount” of a compound of the inventionis meant a sufficient amount of the compound to treat the disorder(e.g., bacterial infection). The specific therapeutically effectiveamount that is required for the treatment of any particular patient ororganism (e.g., a mammal) will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound or composition employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts (see, for example, Goodman and Gilman's, “ThePharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman, J.Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which isincorporated herein by reference in its entirety). The therapeuticallyeffective amount for a given situation can be readily determined byroutine experimentation and is within the skill and judgment of theordinary clinician.

As used herein, “189 mg sodium from sodium chloride per 1000 mg ofceftolozane” refers to a ratio of sodium from the sodium chloride toceftolozane active. For example, “189 mg sodium from sodium chloride per1000 mg of ceftolozane” includes, for example, 94.5 mg sodium fromsodium chloride per 500 mg of ceftolozane, as well as, for example,47.25 mg sodium from sodium chloride per 250 mg ceftolozane. Inaddition, “1,000 mg of ceftolozane as ceftolozane sulfate” refers to anamount of ceftolozane sulfate effective to provide 1,000 mg ofceftolozane. “189 mg sodium from sodium chloride” refers to the amountof sodium chloride (e.g., 480 mg) effective to provide 189 mg of sodium.The amount of sodium from sodium chloride per gram of ceftolozaneactivity in a pharmaceutical composition containing ceftolozane sulfate,chloride and sodium chloride can be calculated using the relevantmolecular weights of ceftolozane, ceftolozane sulfate, sodium chlorideand sodium. For example, a composition comprising about 1,147 mgceftolozane sulfate and 189 mg sodium from sodium chloride contains 480mg sodium chloride per 1,000 mg ceftolozane active.

Unless otherwise indicated, as used herein, the term “RelatedSubstances” with respect to HPLC detection refers to all the ceftolozanerelated process impurities and degradation products other thanceftolozane separated and detected by HPLC according to Example 1.Unless otherwise indicated, as used herein, the term “% RelatedSubstances” refers to the % of the total HPLC peak area obtained byExample 1 attributed to all the ceftolozane related process impuritiesand degradation products other than ceftolozane.

EXAMPLES Example 1: HPLC Analysis of Compositions Comprising Ceftolozane

The purity of ceftolozane in the pharmaceutical compositions wasmeasured using the analytical HPLC method described below.

The HPLC methodologies described herein were used to acquire the dataprovided in Examples 5 and 8.

Analytical HPLC Method

A. Operative Conditions

-   Column: Develosil ODS-UG-5; 5 μm, 250×4.6 mm (Nomura Chemical,    Japan)-   Wavelength: 254 nm (determination of CXA-101 impurities) 210 nm    (determination of Tazobactam Related Compound A)-   Flow rate: 1.0 mL/min-   Column Temperature: 45° C.-   Injection volume: 10 μL-   Run Time: 85 minutes-   Gradient Profile:

Time (min) A % B % 0 75 25 30 70 30 60 0 100 85 0 100 85.1 75 25 110 7525

-   Mobile phase A: Sodium Perchlorate Buffer Solution (pH 2.5) (also    referred to herein as “50 mM Sodium Perchlorate Monohydrate in    water, adjusted to pH 2.5 with Diluted Perchloric Acid”    -   Preparation: Dissolve 7.02 g of Sodium Perchlorate Monohydrate        in 1000.0 mL of water and adjust to pH 2.5 with Diluted        Perchloric Acid (1 in 20).-   Mobile phase B: Sodium Perchlorate Buffer Solution (pH 2.5):    Acetonitrile in a ratio of 4:1 (v/v) (also referred to herein as “63    mM Sodium Perchlorate Monohydrate in water, adjusted to pH 2.5 with    Diluted Perchloric Acid: Acetonitrile, 4:1 (v/v)”)    -   Preparation: Dissolve 8.78 g of Sodium Perchlorate Monohydrate        in 1000.0 mL of water and adjust to pH 2.5 with Diluted        Perchloric Acid (1 in 20). Mix the resulting solution with        Acetonitrile in the ratio 4:1 (v/v).        B. System Suitability Solution (1%)

Transfer 33 mg, accurately weighed, of Sample (use first sample if moreare present) into a 10.0 mL volumetric flask. Dissolve and dilute tovolume with water. Into a 100.0 mL volumetric flask, transfer 1.0 mL ofthis Solution and dilute with water to volume and mix.

C. Sample Preparation.

Transfer 33 mg, accurately weighed, of Sample into a 10.0 mL volumetricflask. Dissolve and dilute to volume with water. Prepare just beforeinjection into the HPLC system.

D. HPLC Analysis Procedure

-   1. Inject Blank (water)-   2. Inject System Suitability Solution and check for tailing factor    and theoretical plates number for CXA-101 and Tazobactam peaks:    -   The tailing factor must not be greater than 1.5    -   Theoretical plates number must not be less than 10000-   3. Inject Sample Solution-   4. Identify the peaks of CXA-101 Related Substances in the Sample    chromatogram, recorded at 254 nm, on the basis of the RRT values    reported in Table 1.-   5. Identify the peak of Tazobactam Related Compound A in the Sample    chromatogram, recorded at 210 nm, on the basis of the RRT values    reported in Table 1A.

TABLE 1A Relative retention times of ceftolozane related substancesImpurity RRT Peak 1 (P1) ~0.15 Peak 2 (P2) ~0.16 Peak 3 (P3) ~0.4 Peak 4(P4) ~0.6 Peak 5 (P5) ~0.89 CXA-101 1.0 Peak 6 (P6) ~1.2 Peak 7 (P7)~1.3 Peak 8 (P8) ~1.4 Peak 9 (P9) ~1.7 Peaks 10, 11 (P10, P11) ~2.3

TABLE 1B Related Substances of CXA-101 Drug Substance and Drug ProductProposed Compound Proposed Structure Source Peak 1 3-side chain

Degradation product and process impurity Peak 2 Unidentified Processimpurity Peak 3 Unidentified Process impurity Peak 4 UnidentifiedProcess impurity Peak 5 7-Epimer type

Degradation product and process impurity Peak 6 NA Process impurity Peak7 Δ3 Isomer type

Degradation product and process impurity Peak 8 Unidentified Processimpurity Peak 9 Anti-Isomer type

Process impurity and Degradation impurity Peaks 10, 11 UnidentifiedProcess impurity

TABLE 1C Relative retention time of tazobactam related compound AImpurity RRT Related Compound A 0.34 Tazobactam 1.0E. Calculations

-   Only peaks with a value ≥LOQ are integrated.    I. CXA-101 Mpurities Determination at 254 nm.-   (Tazobactam and Tazobactam Related compound A peaks are integrated    separately, see below)-   Each related substance at 254 nm is reported for its amount as    expressed by area percent.

$C_{i} = \frac{A_{i} \times 100}{A_{t} + {\Sigma\; A_{i}}}$

where:

C_(i)=Amount of related substance i in the Sample calculated at 254 nm,area %

A_(i)=Peak area of related substance i in the Sample chromatogram

A_(t)=Area of CXA-101 peak in the Sample chromatogram

A_(t)+ΣA_(i)=Total peaks area in the Sample chromatogram

Consider as any Unspecified Impurity, each peak in the chromatogramexcept CXA-101, peaks from 1 to 11 and every peak present in the blankchromatogram and report the largest.

II. Total CXA-101 Related Impurities at 254 nm:

Report the total impurities content as expressed by the followingformula:

$C_{T\; 254} = \frac{\Sigma\; A_{i} \times 100}{A_{t} + {\Sigma\; A_{i}}}$

wherein:

C_(T254)=total impurities content in the Sample calculated at 254 nm,area %

A_(t)=area of CXA-101 peak in the sample chromatogram

ΣA_(i)=total peak areas of impurities in the sample chromatogram

A_(t)+ΣA_(i)=total peaks area in the sample chromatogram

III. Tazobactam Related Compound A Determination at 210 nm:

-   Integrate only the Tazobactam and Tazobactam Related Compound A    peaks

$C_{a} = \frac{A_{a} \times 100}{A_{a} + A_{T}}$

where:

C_(a)=Amount of Tazobactam Related Compound A in the Sample, are %

A_(a)=Peak area of Tazobactam Related Compound A in the Samplechromatogram

A_(t)=Area of Tazobactam in the Sample chromatogram

IV. Total Impurities

-   Make the sum of Tazobactam Related Compound A (III) and the Total    CXA-101 impurities determined (II)    V Quantitation Limit

Component LOQ % Tazobactam 0.03 CXA-101 0.03

Example 2: Screening of Stabilizing Agents

Nine stabilizing agents were screened, including sodium chloride,fructose, xylitol, sorbitol, dextran 40, lactose, glucose, maltose, andD-mannitol. The purity of the ceftolozane in a composition comprising100 mg ceftolozane and 100 mg of one of the stabilizing agents after 3days at 70° C. was compared to a composition comprising 100 mgceftolozane but no stabilizing agent.

As shown in Table 2A, the ceftolozane compositions comprising sodiumchloride, dextran 40, lactose, or maltose were demonstrated to be morestable than the other ceftolozane compositions comprising the otherstabilizing agents, or no stabilizing agent. Sodium chloride and maltosewere selected for further investigation.

TABLE 2A Screening of Stabilizing Agents Stabilizing agent Sodiumchloride Fructose Xylitol Sorbitol Dextran 40 Storage Initial 70° C.Initial 70° C. Initial 70° C. Initial 70° C. Initial 70° C. 3 days 3days 3 days 3 days 3 days Appearance White Pale White Orange WhiteOrange White Pale White Pale mass yellow mass paste mass paste massyellow mass yellow mass paste mass Color and Pale Pale Pale Orange PaleOrange Pale Yellow Pale Yellow clarity yellow yellow yellow and yellowand yellow and yellow and and and and clear and clear and clear andclear clear clear clear clear clear clear pH 5.58 4.23 6.04 3.81 5.964.18 6.01 4.00 5.60 4.36 Residual rate 100.0 75.7 100.0 4.29 100.0 0.41100.0 0.00 100.0 72.2 (%) Reconstitution 15 30 20 40 15 180<    15 160170 160 time(s) Stabilizing agent No stabilizing agent Lactose GlucoseMaltose D-Mannitol (Control) 70° C. 70° C. 70° C. 70° C. 70° C. StorageInitial 3 days Initial 3 days Initial 3 days Initial 3 days Initial 3days Appearance Pale Pale White Pale White Pale White Pale Pale Paleyellow yellow mass yellow mass yellow mass yellow yellow yellow massmass mass mass mass mass mass Color and Pale Pale Pale Pale Pale PalePale Yellow Pale Yellow clarity yellow yellow yellow yellow yellowyellow yellow and yellow and and and and and and and and clear and clearclear clear clear clear clear clear clear clear pH 5.86 4.70 6.23 4.326.08 5.06 6.13 3.97 5.10 4.02 Residual rate 100.0 80.5 100.0 37.3 100.080.9 100.0 1.38 100.0 51.2 (%) Reconstitution 15 15 15 15 15 15 30 50 1515 time(s)

The stabilizing effect of other non-reducing sugars such as sucrose andtrehalose, as well as polyvinylpyrrolidone (PVP), was also evaluated ina ceftolozane formulation.

Five samples were prepared, the components of which are shown in Table2B below. Each sample contained 1000 mg of ceftolozane active, 40 mgcitric acid monohydrate (equivalent of 36 mg citric acid anhydrous), andthe same amount of L-arginine. Stabilizing reagents in four samples are480 mg sodium chloride, 300 mg of trehalose, 300 mg of sucrose, and 300mg of PVP, respectively. One sample was a control that contained nostabilizing reagent. The samples were in lyophilized form and stored at60° C. for 7 days. The purities of the samples were monitored by HPLC onday 0, day 1, day 3 and day 7.

TABLE 2B Comparison between stabilizing reagents Excipient NaClTrehalose Sucrose PVP None Ceftolozane 1000 1000 1000 1000 1000 amountExcipient 480 300 300 300 N/A amount Purity: t₀ 98.42 98.09 98.14 97.8997.94 60° C./1 d 97.85 96.73 96.97 96.05 96.15 60° C./3 d 97.21 95.3695.81 94.57 94.53 60° C./7 d 95.65 94.21 94.19 92.78 92.06 Purity Δ−2.77% −3.88% −3.95% −5.11% −5.88% (0-7 d)

As shown in Table 2B, the sample containing sodium chloride exhibitedthe best stability. The purity of ceftolozane in the sample containingsodium chloride had the slightest purity drop over 7 days. Thisexperiment further supports the discovery that sodium chloride providessurprisingly better stabilizing effect than the other reagents.

Example 3: Stability Study of Ceftolozane Compositions Comprising SodiumChloride, or Maltose, or No Stabilizing Agent

Three ceftolozane compositions were prepared, the components of whichare shown in Table 3. These compositions were put in a stressedstability study at 70° C. for 3 days and 6 days. The purity of theceftolozane in the compositions was analyzed using the HPLC methoddescribed in Example 1.

TABLE 3 Ceftolozane Compositions CEF/no stabilizer 9.5 g activeCeftolozane 5.7 g L-Arginine 200 mg Citric acid CEF/maltose 9.5 g activeCeftolozane 5.7 g L-Arginine 200 mg Citric acid 5 g Maltose H₂OCEF/sodium chloride 9.5 g active Ceftolozane 5.7 g L-Arginine 200 mgCitric acid 4.6 g Sodium Chloride

The results are shown in Table 4 where only the most significantcomposition peaks (P1, P7, and P12) are shown. It was found that thecomposition comprising maltose (CEF/maltose) contained a significantlylarge amount of the composition P12 peak, which was identified as havingthe following formula:

In addition, the presence of maltose produced a particularly aggregatedpowder after lyophilization, which has a potentially negative impact tomanufacturing ceftolozane compositions.

In contrast, the ceftolozane composition comprising sodium chloride(CEF/sodium chloride) was much more stable than the ceftolozanecomposition comprising maltose or the ceftolozane composition comprisingno stabilizing agent. Therefore, sodium chloride was, unexpectedly, abetter stabilizing agent for ceftolozane compositions.

TABLE 4 Stability Study of Ceftolozane Compositions Comprising SodiumChloride, or Maltose, or No Stabilizing Agent P1 P7 P12 Total CEF/nostabilizer Time (days) 0 0.49 0.69 0.00 1.98 3 3.06 1.29 0.00 8.48 64.11 1.49 0.00 10.83 CEF/maltose Time (days) 0 0.41 0.65 0.15 1.91 32.85 1.02 3.44 10.08 6 3.45 1.12 4.01 11.65 CEF/sodium chloride Time(days) 0 0.20 0.62 0.00 1.64 3 1.70 0.85 0.00 4.29 6 2.86 1.05 0.00 6.70

Example 4a: Manufacturing Procedure of Mono Product for Injection

4a.1. Preparation of the Compound Solution of CXA-101 LyophilizedProduct

1) Weigh 30 kg of water for injection into the compounding vessel;

2) Add 100 g of citric acid, anhydrous and 150 g of sodium bicarbonateinto the compounding vessel and dissolve them with mixing;

3) Weigh 5,000 g potency of CXA-101 drug substance and suspend it withmixing. (Note any generation of carbon dioxide.)

4) Slowly add 1,100 g of sodium bicarbonate and dissolve CXA-101 withmixing. (Again, note any generation of carbon dioxide.)

5) Add 1,146 g of sodium chloride and 10,000 g of maltose, dissolve withmixing.

6) Purge dissolved carbon dioxide in the solution with nitrogen untilthe pH of the solution does not change.

7) Adjust the pH of the solution to 6.0±0.1 with 5%-sodium bicarbonatesolution.

8) Adjust the total weight to 56,850 g (D₂₀=1.137) with water forinjection.

9) Confirm the pH of the compounded solution within the range 6.0±0.1.

4a.2. Prefiltration and Sterile-Filtration

10) Filtrate the compounded solution with a sterile tilter-set whichconsists of a 0.2 um polyvinylidene fluoride membrane filter (DURAPORE®,Millipore) and a 0.1 urn polyvinylidene fluoride membrane filter(DURAPORE®, Millipore) connected in tandem. Confirm the integrity ofeach filter before and after the filtration. Take approximately 100 mLof the filtrate in order to check bioburden.

11) Filter the prefiltered compounded solution through a sterilefilter-set which consists of a 0.2 um polyvinylidene fluoride membranefilter and a 0.1 urn polyvinylidene fluoride membrane filter connectedin tandem, and introduce the final filtrate into an aseptic room.Confirm the integrity of each filter before and after the filtration.

4a.3. Processing of container, Stopper and Flip-off Cap

12) Wash a sufficient quantity of 28 mL containers with water forinjection and sterilize the washed containers by a dry-heat sterilizer.Then transfer the sterilized containers into a Grade A area located inan aseptic room.

13) Wash a sufficient quantity of stoppers with, water for injection.Sterilize and dry the washed stoppers by steam sterilizer. Then transferthe sterilized stoppers into a Grade A area located in an aseptic room.

14) Sterilize a sufficient quantity of flip-off caps by steamsterilizer. Then transfer the sterilized flip-off caps into a Grade A orB area located in an aseptic room.

4a.4. Filling and Partially Stoppering

15) Adjust the fill weight of the filtered compounded solution to 11.37g (corresponds to 10 mL of the compounded solution), then start fillingoperation. Check the filled weight in sufficient frequency and confirmit is in target range (11.37 g±1%, 11.26 to 11.43 g). When deviationfrom the control range (11.37 g±2%, 11.14 to 11.59 g) is occurred,re-adjust the filling weight.

16) Immediately after a container is filled, partially stopper thecontainer with a sterilized stopper. Load the filled and partiallystoppered containers onto the shelves of a lyophilizer aseptically.

4a.5. Lyophilization to Crimping, Visual Inspection, Labeling andPackaging

17) After all filled and partially stoppered containers are loaded intoa lyophilizer, start the lyophilization program. Freeze the loadedcontainers at −40° C. and keep at temperature until all containersfreeze. Forward the program to primary drying step (shelf temperature;−20° C., chamber pressure; 100 to 150 mTorr). Primary drying time shouldbe determined by monitoring the product temperature. Forward the programto secondary drying step (shelf temperature; 30° C., chamber pressure;not more than 10 mTorr) after completion of the primary drying step.After all containers are dried completely, return the chamber pressureto atmospheric pressure with sterilized nitrogen. Then stoppercontainers completely.

Example 4b: Manufacturing Procedure of Bulk (Tray) LyophilizedCeftolozane

There are four main steps in the manufacture of a CXA-101 pharmaceuticalcomposition: dissolution, sterile filtration, bulk lyophilization, andpackaging into Sterbags®. These four main steps are composed of a totalof 19 minor steps. The CXA-101 bulk drug product manufacturing processis presented below.

I Dissolution

1. The prescribed amount of water for injection (“WFI”) is charged intothe dissolution reactor.

2. A prescribed amount of citric acid is added.

3. The solution is cooled at 5° C. to 10° C.

4. A prescribed amount of CXA-101 drug substance is added to thesolution.

5. A prescribed amount of L-arginine is slowly added to the solution.

6. A check for complete dissolution is performed. Solution pH isverified to be in the target range of 6.5 to 7.0.

7. A prescribed amount of sodium chloride is added to the solution,wherein the preferred amount of sodium chloride is 125-500 mg of sodiumchloride per 1000 mg of ceftolozane active.

8. A check for complete dissolution is performed. Solution pH isverified to be in the target range of 6.0 to 7.0. If the pH is out ofthis range adjust with either L-Arginine or citric acid.

9. WFI is added to bring the net weight to adjust compounded solutionconcentration.

10. Samples are withdrawn for testing of final pH.

H. Sterile Filtration

11. The solution is passed through the filter (pore size 0.45 μm)followed by double filters (pore size 0.22 μm) onto a shelf on theCriofarma lyophilizer.

12. The line is washed with WFI.

13. The washing solution is passed from Step 12 through sterilefiltration.

III Bulk Lyophilization

14. The washing solution is loaded onto a separate shelf in thelyophilizer (and later discarded).

15. The solution is lyophilized until dry.

16. The product shelf is cooled to 20° C.±5° C.

IV. Packaging into STERBAGS®

17. The lyophilized bulk drug product powder is delumped.

18. The milled powder is sieved.

19. The powder is then discharged into STERBAGS®

Example 5: Stabilizing Effect of Sodium Chloride in CXA-101 Compositions

A. Improvement in the Purity of the Ceftolozane in CXA-101Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

A stability study was carried out at 30° C. and 60° C. and analyzed byHPLC. The sodium chloride content in the CXA-101 compositions isdescribed in Table 5. The HPLC data are summarized in Tables 6-8. Thedata are also plotted in FIGS. 3-6 to show the trends of the purity, andthe amounts of the composition peak 1, the composition with a RRT of0.43 and the composition peak 3, and the composition peak 7 in theCXA-101 compositions with respect to NaCl.

TABLE 5 Sodium Chloride Content in the CXA-101 Compositions Samples NaClcontent A1 481.0 mg NaCl per 1000 mg of ceftolozane A2 190.0 mg NaCl per1000 mg of ceftolozane A3 125.0 mg NaCl per 1000 mg of ceftolozane A475.0 mg NaCl per 1000 mg of ceftolozane A5 50.0 mg NaCl per 1000 mg ofceftolozane

TABLE 6 Purity of Ceftolozane in CXA-101 Compositions with VaryingAmounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 96.6 98.0 97.997.8 97.7 t0/30° C. 0 98.1 97.8 97.8 97.7 1 day/60° C. 1 95.9 96.9 96.595.7 95.5 1 day/30° C. 1 98.2 97.7 97.7 97.6 3 days/60° C. 3 94.9 95.794.8 93.9 93.6 (Δ_(t0-t3)) (1.7) (2.3) (3.1) (3.9) (4.1) 3 day/30° C. 398.0 97.5 97.5 97.3 7 days/60° C. 7 93.6 94.0 94.2 92.3 91.9 7 day/30°C. 7 97.8 97.2 97.1 97.0 Total Δ/60° C. 3.07 4.06 3.7 5.48 5.83 TotalΔ/30° C. 0.3 0.6 0.7 0.7

TABLE 7 HPLC Peak Area of Composition Peak 1 in CXA-101 Compositionswith Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 00.95 0.31 0.3 0.36 0.39 t0/30° C. 0 0.47 0.36 0.36 0.39 1 day/60° C. 11.36 0.86 0.94 1.36 1.39 1 day/30° C. 1 0.48 0.40 0.42 0.48 3 days/60°C. 3 1.71 1.31 1.73 2.06 2.1 3 day/30° C. 3 0.53 0.50 0.52 0.58 7days/60° C. 7 2.26 2.14 2.07 2.86 2.93 7 day/30° C. 7 0.62 0.63 0.660.72 INCREASE %/60° C. 1.31 1.83 1.77 2.5 2.54 INCREASE %/30° C. 0.150.27 0.30 0.33

TABLE 8A HPLC Peak Area of the Composition with a RRT of 0.43 andComposition Peak 3 in CXA-101 Compositions with Varying Amounts ofSodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 0.28 0.10 0.09 0.10 0.11t0/30° C. 0 0.15 0.10 0.10 0.11 1 day/60° C. 1 0.37 0.13 0.16 0.35 0.361 day/30° C. 1 0.13 0.09 0.09 0.10 3 days/60° C. 3 0.68 0.21 0.31 0.710.71 3 day/30° C. 3 0.17 0.13 0.13 0.14 7 days/60° C. 7 1.04 0.36 0.300.81 0.81 7 day/30° C. 7 0.19 0.16 0.16 0.17 INCREASE %/60° C. 0.76 0.260.21 0.71 0.7 INCREASE %/30° C. 0.04 0.06 0.06 0.06

TABLE 8B The HPLC Peak Area of Composition Peak 7 in CXA-101Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5t0/60° C. 0 1.31 0.95 0.96 1.01 1.02 t0/30° C. 0 0.69 1.00 1.01 1.02 1day/60° C. 1 1.37 1.10 1.10 1.23 1.29 1 day/30° C. 1 0.68 0.99 1.01 1.023 days/60° C. 3 1.43 1.19 1.27 1.41 1.46 3 day/30° C. 3 0.68 1.03 1.011.05 7 days/60° C. 7 1.49 1.31 1.35 1.55 1.57 7 day/30° C. 7 0.68 1.011.03 1.07 INCREASE %/60° C. 0.18 0.36 0.39 0.54 0.55 INCREASE %/30° C.NC 0.01 0.02 0.05

Conclusion:

The stability test demonstrates that high sodium chloride contentenhances stability of CXA-101 Compositions.

The HPLC measurements on day 3 were used to analyze the stability of theCXA-101 compositions.

CXA-101 compositions comprising high amounts of sodium chloride (e.g.,125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to bemore chemically stable than CXA-101 compositions comprising low amountsof sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mgof ceftolozane). Table 6 shows that, by day 3 of heating at 60° C.,sample A1, which has the highest salt concentration, is most stable,i.e., has the lowest Δ_(t0-t3) of all samples. By day 3, the sample withthe lowest salt concentration, A5, has the highest Δ_(t0-t3) indicatingthe most degradation. Overall, A5 has degraded 141% more than A1.Further, Table 6 shows that, by day 3 of heating at 60° C., sample A3,which contains a lower salt concentration within the limits of theinvention at 125 mg, is still significantly more stable than A4, acomposition containing 75.0 mg of the salt. A3 has a Δ_(t0-t3) of 3.1,while A4 has a Δ_(t0-t3) of 3.9, meaning that A4 has degraded 26% morethan A3.

B. Long-term Stability Study of CXA-101 Pharmaceutical Compositions withVarying Amounts of Sodium Chloride

Another stability study was carried out at 5° C. and 25° C. The sodiumchloride content in the CXA-101 compositions is described in Table 9.The amounts of citric acid and L-arginine in each composition were thesame. These samples were in lyophilized form and were placed onlong-term (24-36 months), real time stability programs.

The composition peak 1 is considered “diagnostic” for formulationfailure because it is the first peak to go out of trend or specification(1.5%). Thus, the stability of these CXA-101 compositions was alsomeasured by the length of storage until formulation failure as indicatedby the composition peak 1. The data in Table 9 were extrapolated fromdata collected after 4 months. Clearly, based on the amount of thecomposition peak 1 in the compositions, the composition with about 480mg sodium chloride per 1 gram ceftolozane active was significantly morestable than the compositions containing 125 mg or 62.5 mg sodiumchloride per 1 gram of active ceftolozane (i.e., stability ofceftolozane compositions: 480>>125 mg>62.5 mg).

TABLE 9 The Peak 1 Failure Points of CXA-101 Compositions with VaryingAmounts of Sodium Chloride Peak 1 failure Peak 1 failure Ceftolozaneactive, 1 g + point at 5° C. point at 25° C. 480 mg NaCl 245 months 15months 125 mg NaCl 70 months 5 months 62.5 mg NaCl 25 months 3 months**Results at 3 months = 1.34%, 4 months = 1.15%

Example 6: Manufacturing Process of a CXA-201 Composition ComprisingTazobactam and CXA-101/Ceftolozane by Co-Lyophilization

The manufacturing process of a CXA-201 composition comprising tazobactamand ceftolozane by co-lyophilization is shown in FIG. 2. Non-sterilebulk tazobactam and bulk ceftolozane were mixed, followed by dissolutionand sterile filtration. The filtrate was then tray-lyophilized to obtainthe CXA-201 composition. The CXA-201 composition can be container-filledas a final drug product. The components of a CXA-201 compositionprepared by co-lyophilization are shown in Table 10.

TABLE 10 Components of a CXA-201 Composition Prepared byCo-lyophilization Component Function Amount (mg/container) CeftolozaneActive pharmaceutical 1000 (potency) ingredient L-arginine Alkalizationreagent 587 Citric acid (anhydrous) Buffer 21 Sodium chloride Stabilizer476 Tazobactam (free acid) Active pharmaceutical 500 ingredient Sodiumbicarbonate Alkalization reagent Quantity sufficient¹ for pH 4.8 to 7.0Water Dissolution solvent Not more than 4% by weight² Nitrogen Inert gasSufficient quantity ¹Sodium content is approximately 78 mg/g oftazobactam in drug product after lyophilization. ²Water is removedduring the lyophilization process and is controlled at no more than 4%by weight.

Example 7a: Assessment of Co-Lyophilized Combo Drug Product (i.e., aCXA-201 Composition)

A. Preparation of the Co-Lyophilized Combo Drug Product (i.e. theCXA-201 Composition)

The components of the co-lyophilized CXA-201 composition are shown inTable 11. This composition was prepared, as described above in Example6.

TABLE 11 Components of the CXA-201 Composition Prepared byCo-Lyophilization CXA-201 16.3 g active ceftolozane Comp. 8.1 g activeTazobactam free ac. 15.5 g L-Arginine 350 mg Citric acid 7.9 g NaCl 6.1pH compounded solutionB. Stressed Stability Test

Stability studies of this CXA-201 composition prepared byco-lyophilization were carried out at 25° C. and 40° C. The compositionwas analyzed using HPLC. The following Tables 12 and 13 are summaries ofthe HPLC measurements at time zero, after one month (T1), and afterthree months (T2).

TABLE 12 Stability Data of Co-Lyophilized CXA-201 Composition at 25°C./RH = 60% Test items Spec. D.P. T0 T1 25° C. T2 25° C. RelatedSubstances Peak1 ≤1.50% 0.31% 0.54% 0.71% Peak2 ≤0.40% 0.07% 0.07% 0.09%Peak3 ≤0.30% <0.03% <0.03% <0.03% Peak4 ≤0.80% 0.08% 0.08% 0.09% Peak5≤1.00% 0.27% 0.26% 0.29% Peak6 ≤0.15% <0.03% <0.03% <0.03% Peak7 ≤2.00%0.64% 0.65% 0.66% Peak8 ≤0.15% <0.03% <0.03% <0.03% Peak9 ≤0.60% 0.05%0.11% 0.10% Peak10, 11 ≤0.15% 0.04% 0.04% 0.04% each Peak12 ≤2.00%<0.03% <0.03% <0.03% Others (RRT 0.43) ≤0.15% <0.03% <0.03% 0.04% Others(RRT 1.22) ≤0.15% 0.13% 0.30% 0.38% Others (RRT 2.18) ≤0.15% 0.03%<0.03% 0.05% Others (RRT 2.77) ≤0.15% <0.03% 0.03% 0.03% Sing. Unk.≤0.15% 0.05% 0.07% 0.05% Total ≤5.00% 1.67% 2.19% 2.77% pH report value5.5 4.83

TABLE 13 Stability Data of Co-Lyophilized CXA-201 Composition at 40°C./RH = 75% Test items Spec. D.P. T0 T1 40° C. T2 40° C. RelatedSubstances Peak1 ≤1.50% 0.31% 1.77% 2.22% Peak2 ≤0.40% 0.07% 0.10% 0.16%Peak3 ≤0.30% <0.03% <0.03% 0.06% Peak4 ≤0.80% 0.08% 0.09% 0.09% Peak5≤1.00% 0.27% 0.27% 0.30% Peak6 ≤0.15% <0.03% <0.03% <0.03% Peak7 ≤2.00%0.64% 0.69% 0.78% Peak8 ≤0.15% <0.03% <0.03% 0.10% Peak9 ≤0.60% 0.05%0.09% 0.09% Peak10, 11 ≤0.15% 0.04% 0.04% 0.05% each Peak12 ≤2.00%<0.03% <0.03% <0.03% Others (RRT 0.43) ≤0.15% <0.03% 0.09% 0.15% Others(RRT 1.22) ≤0.15% 0.13% 0.74% 0.97% Others (RRT 2.18) ≤0.15% 0.03%<0.03% 0.08% Others (RRT 2.77) ≤0.15% <0.03% <0.03% 0.04% Sing. Unk.≤0.15% 0.05% 0.11% 0.25% Total ≤5.00% 1.67% 4.49% 6.32% pH report value5.5 4.09C. Conclusion:

A new compound having RRT=1.22 was observed in the co-lyophilizedCXA-201 compositions. While not wishing to be bound by theory, thecompound RRT 1.22 was identified as a compound formed by a reactionbetween ceftolozane and formylacetic acid, which was a by-product oftazobactam as illustrated in Marunaka et al. (Chem. Pharm. Bull. 1988,Vol. 36 (11), pp. 4478-4487). The stability data at 25° C. and at 40° C.have confirmed the continued formation of the compound RRT 1.22 over thecourse of time.

Example 7b: Identifying the Compound of Formula (III)

The Co-Lyophilized Combo Drug Product was prepared as described above inExample 6. The formulation composition of the Co-Lyophilized Combo drugproduct is shown in Table 11 (Example 7a). This sample maintained at 25°C./RH=60% and 40° C./RH=75% after one month (T1) and three months (T2).Samples were analyzed using a HPLC method as described in Example 1. Thedata for analysis of the samples by HPLC is shown in Example 10 in Table23 (Stability data of Co-Lyo Combo Drug Product at 25° C.) and Table 24(Stability data Co-Lyo Combo Drug Product at 40° C.). The presence ofthe compound of Formula (III) was identified has having a retention timeof about 1.22 as measured by HPLC (see Example 2). RRT=1.22 was observedin co-lyophilized drug product. The compound of formula (III) isbelieved to be formed by a reaction between ceftolozane and formylaceticacid, which was a degradation product of tazobactam. The amount of thecompound of formula (III) in a composition comprising ceftolozane andtazobactam can be increased over time at 25° C. and at 40° C.

The material obtained from the RRT 1.22 peak was analyzed by LC/MS,providing the spectra shown in FIG. 14. FIG. 15 (below) is thecorresponding structures for the peaks shown in FIG. 14.

A test sample prepared from ceftolozane and tazobactam acidco-compounding solution containing RRT 1.22 impurity was used on theLC/MS experiment. Liquid chromatography separation was performed on aZorbax SB C8, 3.5 μm, 3.0 mm×150 mm column, using gradient elution with20 mM ammonium formate containing 0.1% Heptofluorobutyric acid pH 3.2 asmobile phase A and 0.1% Heptofluorobutyric acid in acetonitrile asmobile phase B. The gradient starts from 3% (initial) to 15% mobilephase B in 20 minutes (with RRT 1.22 eluting at about 10.7 minutes).Mass detection was performed using electrospray ionization techniqueunder positive mode. The column effluent was also monitored at 254 nmusing a photodiode-array detector. MS/MS fragmentation was performed onm/z 737.3 positive ion using nitrogen as collision gas, with collisionenergy set at 35V.

Example 8: Stabilizing Effect of Sodium Chloride in CXA-201 Compositions

A. Reduction of the Composition at RT=63 Minutes in CXA-201 Compositions

A stability study was carried out at 25° C. and analyzed by HPLC.CXA-201 compositions comprise ceftolozane and tazobactam, furthercomprising high, mid, or low amounts of sodium chloride (480, 125, or62.5 mg NaCl per 1000 mg of ceftolozane, respectively). Comparison ofthe compositions are listed in Table 14. The amounts of the compositionRT 63′, as measured by the HPLC method, are summarized in Table 15.

TABLE 14 Comparison of the CXA-201 Compositions Lot CXA-101 NaClTazobactam C1 10% High Na C2 20% Mid Na C3 20% Low Na C4 20% MidArginate C5 20% Low Arginate

TABLE 15 RT 63′ Peak Area at t = 3 months, 25° C./60% RH storage 1^(st)data 2nd data 3rd data collection collection collection Sample SummaryRT Area % RT Area % RT Area % C1 High salt + 63.90 0.03 63.30 0.08 62.490.14 Tazo Na C2 Mid salt + 63.78 0.06 63.12 0.12 62.45 0.28 Tazo Na C3Low salt + 63.75 0.12 63.11 0.14 62.46 0.29 Tazo Na C4 Mid salt + 63.760.10 63.16 0.13 62.44 0.28 Tazo Arg C5 Low salt + 63.72 0.08 63.14 0.1662.46 0.33 Tazo ArgConclusion:

At the three month time point, the reduced salt formulations wereobserved to be not as stable as the full salt formulation; and trendsindicate that reduction in salt causes at least 1.5-fold greatercomposition at RT=63 minutes, as measured by HPLC. The compositionscomprising 480 mg NaCl per 1000 mg of ceftolozane had the least amountof the composition RT 63′ after 3 months at 25° C. The amount of thecomposition RT 63′ in the compositions comprising 125 mg NaCl per 1000mg of ceftolozane was 1.5-fold or greater than the amount of thecomposition 63′ in the compositions comprising 480 mg NaCl per 1000 mgof ceftolozane. The amount of the composition RT 63′ in the compositionscomprising 62.5 mg NaCl per 1000 mg of ceftolozane was 2-fold or greaterthan the amount of the composition RT 63′ in the compositions comprising480 mg NaCl per 1000 mg of ceftolozane. Thus, the CXA-201 compositionscomprising high amounts of sodium chloride (e.g., 125-1000 mg sodiumchloride per 1000 mg of ceftolozane) were more chemically stable thanthe compositions comprising low amounts of sodium chloride (e.g., lessthan 125 mg sodium chloride per 1000 mg of ceftolozane).

B. Improvement in the Purity of Ceftolozane in CXA-201 PharmaceuticalCompositions with Varying Amounts of Sodium Chloride

A stability study was carried out at 30° C. and 60° C. analyzed by HPLC.The sodium chloride content in the CXA-201 compositions is described inTable 16. The HPLC data at 60° C. are summarized in Tables 17-20. Thedata are also plotted in FIGS. 7-10 to show the trends of the purity,and the amounts of the composition peak 1, the composition with a RRT of0.43 and the composition peak 3, and the composition peak 7 in theCXA-201 compositions with respect to NaCl.

TABLE 16 The Sodium Chloride Content in the CXA-201 Compositions SamplesNaCl content B1 481.0 mg sodium chloride per 1000 mg of ceftolozane B2125.0 mg sodium chloride per 1000 mg of ceftolozane B3 75.0 mg sodiumchloride per 1000 mg of ceftolozane B4 50.0 mg sodium chloride per 1000mg of ceftolozane

TABLE 17 The Purity of Ceftolozane in CXA-201 Compositions with VaryingAmounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 98.1 97.8 97.8 97.7 1day/60° C. 1 97.2 96.3 96.2 96.0 1 day/30° C. 1 98.2 97.7 97.6 97.6 3days/60° C. 3 95.4 94.9 94.7 94.6 (Δ_(t0-t3)) (2.7) (2.9) (3.1) (3.1) 3day/30° C. 3 98.0 97.5 97.4 97.3 7 days/60° C. 7 92.7 93.8 93.6 93.4 7day/30° C. 7 97.8 97.2 97.0 96.9 Total Δ/60° C. 5.3 4.0 4.2 4.3 TotalΔ/30° C. 0.3 0.6 0.8 0.8

TABLE 18 The HPLC Peak Area of Composition Peak 1 in CXA-201Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t00 0.47 0.38 0.38 0.41 1 day/60° C. 1 1 1.08 1.09 1.14 1 day/30° C. 10.48 0.44 0.45 0.49 3 days/60° C. 3 1.85 1.64 1.66 1.71 3 day/30° C. 30.53 0.53 0.56 0.61 7 days/60° C. 7 3.3 2.28 2.25 2.29 7 day/30° C. 70.62 0.67 0.71 0.77 INCREASE %/60 ° C. 2.83 1.9 1.87 1.88 INCREASE %/30° C. 0.15 0.29 0.33 0.36

TABLE 19 The Total HPLC Peak Area of the Composition with a RRT of 0.43and Composition Peak 3 in CXA-201 Compositions with Varying Amounts ofSodium Chloride Day B1 B2 B3 B4 t0 0 0.15 0.12 0.12 0.12 1 day/60° C. 10.36 0.35 0.31 0.32 1 day/30° C. 1 0.13 0.12 0.13 0.12 3 days/60° C. 30.92 0.67 0.65 0.62 3 days/30° C. 3 0.17 0.16 0.17 0.16 7 days/60° C. 71.29 0.78 0.75 0.71 7 days/30° C. 7 0.19 0.19 0.20 0.20 INCREASE %/60 °C. 1.14 0.66 0.63 0.59 INCREASE %/30 ° C. 0.04 0.07 0.08 0.08

TABLE 20 The HPLC Peak Area of Composition Peak 7 in CXA-201Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t00 0.69 1.01 1.01 1.01 1 day/60° C. 1 0.73 1.12 1.15 1.18 1 day/30° C. 10.68 1.00 0.99 0.95 3 days/60° C. 3 0.8 1.24 1.27 1.27 3 days/30° C. 30.68 1.00 1.01 1.03 7 days/60° C. 7 0.94 1.32 1.35 1.4 7 days/30° C. 70.68 1.02 1.05 1.06 INCREASE %/60° C. 0.25 0.31 0.34 0.39 INCREASE %/30°C. NC 0.01 0.04 0.05Conclusion:

The stability data shows that high sodium chloride content enhancesstability of CXA-201 compositions.

Similarly to CXA-101 compositions, CXA-201 compositions comprising highamounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000mg of ceftolozane) were found to be more chemically stable than CXA-201compositions comprising low amounts of sodium chloride (e.g., less than125 mg sodium chloride per 1000 mg of ceftolozane). Table 17 shows that,by day 3 of heating at 60° C., sample B1 containing the highest saltconcentration is most stable, i.e., has the lowest Δ_(t0-t3) of allsamples. By day 3, the sample with the lowest salt concentration, B4,has the highest Δ_(t0-t3) indicating the most degradation. Overall, B4has degraded 15% more than B1.

Example 9: Manufacturing Process of a CXA-201 Composition (ComprisingTazobactam and Ceftolozane) by Blending

A. Sterile Dry Blending of Bulk Lyophilized Ceftolozane and BulkLyophilized Tazobactam

A low energy drum blender that agitates the material by tumbling andalso moving the bed up and down is used. A representative process ofblending is described as follows, also shown in FIG. 1. The blender wascharged with 23.4 kg of CXA-101 bulk product, and 5.4 kg of tazobactambulk product. Both the CXA-101 and tazobactam were individuallylyophilized beforehand. The material was blended for 180 minutes.In-process tests of content assay for both CXA-101 and tazobactam wereperformed to assess the homogeneity using the samples of blend materialstaken from three places. The relative standard deviation (RSD) for eachof CXA-101 and tazobactam content assay was no greater than 2% and theRSD for the ratio of CXA-101/tazobactam was no greater than 2% (SeeTable 21).

TABLE 21 In-Process Testing of Blending Samples of a CXA-201 Compositionat Three Places Results Acceptance Limits 60 120 180 Test (expectedvalue) Sampling minute minute minute Content: Ceftolozane¹ 30.4%-37.2% 134.24 34.07 34.42 2 34.62 34.21 34.66 3 34.71 34.60 34.85 Mean³ 34.5234.30 34.64 RSD % 0.72 0.80 0.63 Content: Tazobactam² 15.2%-18.6% 117.96 18.20 17.12 2 16.90 18.26 16.51 3 17.27 16.93 17.02 Mean³ 17.3817.80 16.89 RSD % 3.10 4.22 1.96 Ratio of Content (w/w) 2.00⁴ 1 1.911.87 2.01 ceftolozane/tazobactam 2 2.05 1.87 2.10 3 2.01 2.04 2.05 Mean³1.99 1.93 2.05 RSD % 3.69 5.12 2.2 RSD = relative standard deviation¹Theoretical value: 33.96% Accetance limits are 90%-110% of thetheoretical value. ²Theoretical value: 16.99% Acceptance limits are90%-110% of the theoretical value. ³Three samples are taken at each timepoint at three places to measure the percentage by weight of ceftolozaneand tazobactam. The “Mean” is the average of the percentages or theweight ratios of Ceftolozane/tazobactam. ⁴Acceptance limits wereestablished based on batch history.B. Packaging into STERBAGS®

-   The blended powder is then discharged into STERBAGS®.    C. Finished CXA-201 Drug Product

A fill and finish process is utilized for the final drug product, whichis a pharmaceutical composition comprising CXA-101 and tazobactam at aratio of 1000 mg/500 mg. Glass containers are washed with WFI anddepyrogenated in a Class 100 depyrogenation tunnel at a temperature of320° C. Pre-washed and pre-siliconized stoppers are autoclaved for 40minutes at 121° C. The bulk drug product is packaged in a STERBAG®system comprised of three bags. The outer bag is cleaned withdisinfectant in a Class 10,000 clean room. The bag system is placed in apass-through UV box where it is subjected to UV radiation (>20 μW/cm²)for 20 minutes to sterilize the surface of the outer bag. The outer bagis removed and left in the UV box. The middle bag is placed in a Class Alaminar airflow (LAF) hood. The sterile middle bag is removed under LAF.The sterile, bottle-shaped inner bag is then placed in a sterilestainless steel carrier and attached to the filling machine.

Sterile bulk CXA-101/tazobactam drug product is filled under a nitrogenblanket into 30-mL, Type I clear glass containers. The sterile drugproduct is gravity-fed into the filling machine under LAF. containerfill weights are periodically checked throughout the filling operationto ensure proper operation of the filling line. Filling and stopperingoperations are performed under Class 100 LAF conditions. Capping andcontainer washing are done in the Class 10,000 clean room.

Example 10: Assessment of Blend Combination Drug Product

A. Preparation of the Blend Combination Drug Product (CXA-201Composition)

The blend drug product was prepared, as described above in Example 9, onlab scale using a small blender. The components of the blend compositionare shown in Table 22.

TABLE 22 Components of the Blend Composition Quantity as activeComponent Composition components CXA-201 CXA-101 for Ceftolozane 10.8 gComp. Injection L-Arginine  6.7 g Bulk (25 g) Citric acid  233 mg Sodiumchloride  5.2 g Tazobactam  5.4 g (as Tazo free acid) sodium sterileBulk (6 g)B. Stressed Stability Test

Stability studies of this CXA-201 composition prepared by the blendingprocess were carried out at 25° C. and 40° C. The composition wasanalyzed using HPLC method described in Example 1. The following Tables23 and 24 are summaries of the HPLC measurements at time zero, after onemonth (T1), and after three months (T2).

TABLE 23 Stability Data of Blend CXA-201 Composition at 25 ° C./RH = 60%Test items Specifications T0 T1 25° C. T2 25° C. Related SubstancesPeak1 ≤1.50% 0.61% 0.93% 1.08% Peak2 ≤0.40% <0.03%   <0.03%   <0.03%  Peak3 ≤0.30% <0.03%   <0.03%   <0.03%   Peak4 ≤0.80% 0.03% 0.03% 0.04%Peak5 ≤1.00% 0.09% 0.12% 0.13% Peak6 ≤0.15% <0.03%   <0.03%   <0.03%  Peak7 ≤2.00% 1.28% 1.34% 1.35% Peak8 ≤0.15% <0.03%   <0.03%   <0.03%  Peak9 ≤0.60% 0.03% <0.03%   0.03% Peak10, 11 ≤0.30% <0.03%   0.04% 0.05%Sing. Unk. ≤0.15% 0.13% 0.13% 0.14% Total ≤5.00% 2.49% 3.03% 3.28% AssayCXA-101 Teor. % = 32.6% 32.5% n.a. n.a. Assay Teor. % = 17.4% 18.2% n.a.n.a. Tazobactam Tazobactam  ≤4.0% 0.07% 0.12% 0.14% Related Compound AK.F.  ≤4.0%  2.6% n.a. n.a. pH 5.0-7.0 6.0 5.6 5.1

TABLE 24 Stability Data of Blend CXA-201 Composition at 40° C./RH = 75%Test items Specifications T0 T1 40° C. T2 40° C. Related SubstancesPeak1 ≤1.50% 0.61% 1.66% 2.28% Peak2 ≤0.40% <0.03%   <0.03%   <0.03%  Peak3 ≤0.30% <0.03%   <0.03%   0.04% Peak4 ≤0.80% 0.03% 0.04% 0.05%Peak5 ≤1.00% 0.09% 0.13% 0.14% Peak6 ≤0.15% <0.03%   <0.03%   <0.03%  Peak7 ≤2.00% 1.28% 1.41% 1.46% Peak8 ≤0.15% <0.03%   <0.03%   <0.03%  Peak9 ≤0.60% 0.03% <0.03%   0.03% Peak10, 11 ≤0.30% <0.03%   0.08% 0.09%Sing. Unk. ≤0.15% 0.13% 0.14% 0.13% Total ≤5.00% 2.49% 4.21% 5.27% AssayCXA-101 Teor. % = 32.6% 32.5% n.a. n.a. Assay Teor. % = 17.4% 18.2% n.a.n.a Tazobactam Tazobactam  ≤4.0% 0.07% 0.35% 0.54% Related Compound AK.F.  ≤4.0%  2.6% n.a. n.a. pH 5.0-7.0 6.0 5.0 4.4C. Conclusion

The data at both 25° C. and at 40° C. have shown that the blendingprocess completely inhibits formation of the compound RRT=1.22.

Example 11: Alkalizing Agent Selection

Compositions for intravenous administration should be formulated toresemble the and pH of human blood to reduce vascular complications. Therecommended pH is between 5 and 9 (ideal pH is as close to 7.4 aspossible). Departing from this recommended pH ranges of an intravenouslyadministered composition can result in the development of complicationssuch as phlebitis, or inflammation of the veins. Marc Stranz, A Reviewof pH and Osmolarity, 6 Int'l J. of Pharm. Compounding 216, 218(May/June 2002). Unfortunately, few drug infusions are stable at asuitable pH for intravenous administration. Depending on the molecularstructure, a drug is most stable or has the best solubility at aparticular pH range (e.g., pH<6) and divergence from this pH range maylead to increased drug decomposition. It is thus challenging to find abalance between the safe limits of pH and optimum drug stability incompositions for intravenous administration. Marc Stranz, TheImplications of Osmolality, Osmolarity and pH in Infusion Therapy, INSAnnual Conference (May 2005).

A formulation close to physiologic pH was targeted. This necessitates analkalizing agent due to intrinsic pH 1.92 of ceftolozane in solution(2%). The initial study of alkalizing agents included sodium hydroxide,L-arginine, tris, sodium bicarbonate, meglumine, diethanolamine, andtriethanolamine. Samples containing 100 mg ceftolozane sulfate, 22.9 mgsodium chloride, 200 mg maltose, and 2 mg citric acid anhydrous wereprepared and adjusted to ˜pH 4. The samples were lyophilized and powdersstored at 70° C. for 3 days, 60° C. for 3, 6 and 9 days and at 40° C.for one month. The stored samples were then analyzed for ceftolozanecontent. Results are reported below in Table 25:

TABLE 25 Effect of Alkalizing Agent on Ceftolozane Recovery Storage ↓Sodium hydroxide L-arginine Tris Sodium bicarbonate MeglumineDiethanolamine Triethanolamine 70° C., 3 d 93.3 93.0 83.1 93.8 71.2 52.728.0 60° C., 3 d 97.0 96.3 93.5 93.9 94.4 91.6 67.2 60° C., 6 d 95.795.5 89.8 96.0 89.8 83.6 59.0 60° C., 9 d 93.9 93.1 87.5 93.8 88.7 82.075.9 40° C./75% RH, 1 mo 97.3 97.0 95.1 97.6 97.6 94.4 94.4

Ceftolozane recovery was consistently above 90% in the presence ofsodium hydroxide, L-arginine, or sodium bicarbonate. Although sodiumhydroxide performed well, as a strong base, it could promote basehydrolysis of the active more readily during scale up and be moredifficult to dry during lyophilization than other alkalizing agents.Accordingly sodium hydroxide was not considered for further formulationdevelopment. L-arginine was thus chosen as the alkalizing agent for theformulation.

To ensure suitability of L-arginine as an alkalizing agent, a study wasconducted to compare L-arginine against sodium bicarbonate. In thisstudy, solutions were prepared to contain ceftolozane in the presence ofsodium chloride and citric acid adjusted to approximately pH 6 witheither L-arginine or sodium bicarbonate. The solutions were thenlyophilized and samples distributed for accelerated and regular storage.A summary of the total additional compounds and pH for the variousconditions after 1 month is presented in Table 26.

TABLE 26 Effect of L-Arginine and Sodium Bicarbonate on CeftolozaneRelated Substances during Storage, pH 6 Bulk solution composition per1000 mg ceftolozane free base 632 mg L-arginine 288 mg sodium 485 mgsodium bicarbonate chloride 481 mg sodium chloride 21 mg citric acid 21mg citric acid Total Related Sample Total Related Sample Storagecondition Substances pH Substances pH Initial 1.42% 5.8 2.12% 5.8  5°C., 1 month 1.38% 5.8 2.66% 5.6 25° C., 1 month 1.74% 5.5 4.99% 4.8 40°C., 1 month 2.32% 5.0 5.93% 4.5

As seen in the table the bicarbonate-adjusted sample showed a largerincrease in related substances and a less stable pH profile.Accordingly, it was decided to maintain L-arginine as the alkalizingagent in the formulation.

Example 12: Components of a CXA-201 Composition

An example of a batch formulae for ceftolozane composition (compoundingof ceftolozane substance with excipients such as citric acid, sodiumchloride, and L-arginine followed by sterile lyophilization) is foundbelow in Table 27.

TABLE 27 Batch Formula for Ceftolozane composition Amount per TargetComposition Batch (kg) Component mg/g 1 2 Ceftolozane Sulfate^(i)) 172.1114.0 202.6 Citric Acid, Anhydrous, USP 3.2 2.1 3.7 Sodium Chloride, USP73.1 48.3 86.0 L-Arginine, USP ~90 59.7 106.0 QS to achieve targetpH^(ii)) Water for Injection, USP QS to 1000 QS QS Total Batch Size 6601175 ¹⁾Ceftolozane sulfate is charged based on its measured potency toobtain 150 mg free base/g solution. ^(ii))L-arginine is added as neededto obtain pH 6.5 ± 0.5 in the bulk solution; 90 mg per gram solution isconsidered a representative amount.

An example of a batch formula for the ceftolozane/tazobactam drugproduct is presented in Table 28 below.

TABLE 28 Batch Formula Ceftolozane/Tazobactam Drug Product CompositionAmount per Amount per Batch, Component container, mg kg Ceftolozane 2255112.8 composition^(i)) Tazobactam^(ii)) 537 26.9 Nitrogen, NF^(iii)) — —Total 2792 139.7 Total Batch Size, kg 139.7 Total container Quantity50,000 ^(i))The target fill for ceftolozane is 1000 mg free base, addedto the container as the composition. The amount 2255 mg is based on 100%theoretical potency of the composition. Actual weight will vary based oncomposition measured potency. ^(ii))The target fill for tazobactam is500 mg free acid, added to the container as its sodium salt form. Theamount 537 mg is based on 100% theoretical potency. ^(iii))Nitrogen isused as a processing aid to blanket containers after powder filling andprior to insertion of stopper.The unit composition of a dosage for reconstitution is described inTable 29.

TABLE 29 Unit Compositions of Ceftolozane/Tazobactam for Injection, 1000mg/500 mg Nominal Composition Component Function mg per containerCeftolozane Ceftolozane Active 1147 composition¹⁾ Sulfate Citric Acid,Chelating Agent 21 Anhydrous Sodium Stabilizing Agent 487 ChlorideL-Arginine Alkalizing Agent 600²⁾ Q.S. for pH adjustment TazobactamSodium³⁾ Active 537 Nitrogen Processing Aid^((a)) Q.S. Total Weight 2792¹⁾Actual amount of ceftolozane composition will vary based on themeasured potency. Ceftolozane sulfate, 1147 mg, corresponds to 1000 mgceftolozane free base. ²⁾L-arginine is added as needed to achieve pH 6.5± 0.5; 600 mg per container is considered a representative total amount.³⁾Actual weight of tazobactam sodium will vary based on the measuredpotency. Tazobactam sodium 537 mg, corresponds to 500 mg tazobactam freeacid 4) Nitrogen blanket is applied after powders are dispensed to thecontainer and prior to insertion of stopper.

Example 12a: Development and Implementation of a System to PreventCross-Contamination in Accordance with FDA Guidance

A recently published (April 2013) Food and Drug Administration Guidancefor Industry Non Penicillin Beta-Lactam Drugs: A CGMP Framework forPreventing Cross-Contamination provides direction on prevention ofcross-contamination for facilities that manufacture non-penicillinbeta-lactam drugs. Provided herein are steps for the development andimplementation of a system to prevent cross-contamination due to theintroduction of both sterile ceftolozane drug product intermediate andtazobactam sodium into a facility that is in conformance with FDAGuidance.

Segregation steps to conform with FDA Guidance can include, but are notlimited to:

-   -   Relocation all other drug products to other sites    -   Separating the ceftolozane/tazobactam product filling line and        the veterinary cephapirin product filling line    -   Creating separate HVAC systems    -   Establishing separate warehouse areas    -   Formalizing separate material, waste and personnel flows    -   Constructing temporary facilities for gowning and entrance to        the line used for the ceftolozane/tazobactam drug product.    -   Constructing new walls, modifying and reinforcing existing walls    -   Equipping the existing emergency egress with alarms and gaskets        to completely separate both lines throughout all the floors of        the building    -   Creating the permanent separation of locker, rest and break        rooms for both lines of the facility:    -   Dedicated maintenance and operations personnel for each part of        the facility including different uniform colors for each part of        the facility    -   Dedicated equipment and tools for each part of the facility    -   An Emergency Recovery plan

Example 13: Physicochemical and Biological PropertiesCeftolozane/Tazobactam for Injection, 1000 mg/500 mg

As a product intended for intravenous use, several properties areimportant for physiological compatibility. These include particulatematter, sterility, endotoxin limit, pH, and osmolality. Particulatematter and sterility are controlled at the point of manufacture. Thedrug product is processed aseptically throughout the entiremanufacturing process, inclusive of ceftolozane, tazobactam sodium, andceftolozane/tazobactam in-container drug product.

The ceftolozane/tazobactam drug product is controlled to approximatelypH 6, to provide physiological comfort, while still assuring adequatestability for the drug substances. The ceftolozane drug productintermediate is controlled during compounding to pH 6.5±0.5 and iscontrolled at release to pH 5 to 7. The tazobactam sodium is controlledat release to pH 5 to 7.

Ceftolozane/tazobactam following reconstitution with normal saline anddilution for infusion also in normal saline (10 mg/mL ceftolozane; 5mg/mL tazobactam) is slightly hypertonic, with osmolality approximately500 mOsm/kg. However, slightly hypertonic intravenous infusion solutionsare not uncommon as drug products are commonly prepared and diluted withalready-isotonic solutions, such as normal saline. The generallyaccepted maximum upper limit for peripheral intravenous administrationis approximately 900 mOsm/kg, though admixtures 600 to 900 mOsm/kg aretypically administered through a central line. Therefore, to be withinthe limits of this range, the infusion product is less than 600 mOsm/kg.

Example 14: Determining Osmolality of CXA-201 Compositions

-   CXA-101 and Tazobactam Sodium samples (#1-#3) were reconstituted as    follows:-   Sample#1: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of    CXA-101 dissolved in 4 mL of WFI Water and 6 mL of USP Normal    Saline.-   Sample#2: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of    CXA-101 dissolved in 4 mL of WFI Water added 10 mL of USP Normal    Saline.-   Sample#3: Weighed 0.103 g of Tazobactam Sodium dissolved in 1 mL of    WFI Water and 0.462 g of CXA-101 dissolved in 1 mL of WFI Water then    mixed together added 10 mL of USP Normal Saline.-   Tazobactam Sodium (Potency: 97.5%)-   CXA-101 (Potency: 43.3%)-   WFI Water-   USP Normal Saline

The osmolality of CXA-101 and Tazobactam Sodium samples (#1-#3) was thendetermined using a freezing point depression Osmometer (available fromAdvanced Instruments, Inc.).

TABLE 30A Osmolality of Reconstituted Solutions Tazobactam CXA-101Sodium Conc. Conc. Osmolality Sample# (mg/ml) (mg/ml) WFI Water Saline(mOsm/kg) 1 20.0 10.0 40% (40 mL) 60% (60 mL)  589 2 14.3 7.1 29% (40mL) 71% (100 mL) 512 3 16.7 8.3 17% (20 mL) 83% (100 mL) 604

A unit dosage form composition of Table 29 was reconstituted with 10 mLof Sterile WFI or USP Normal Saline then added into 100 mL 5% DextroseInjection (D5W) or 0.9% Sodium Chloride (NS) bags and the osmolality ofthe resulting bag solution was determined as shown in table Table 30Bbelow.

TABLE 30B Osmolarity of Ceftolozane Bag Solution (mOsm/kg) Time PointsWFI-D5W NS-D5W sWFI-NS NS-NS RT T0 446 470 449 478

In Table 30B, data for osmolality of the following productreconstitution scenarios was determined using the composition from Table29

-   -   5% Dextrose Injection USP, 100 mL Bag (Baxter)    -   0.9% Sodium Chloride Injection USP, 100 mL Bag (Baxter)    -   sWFI—D5W: reconstituted with Sterile WFI then added into 5%        Dextrose Injection bag    -   NS—D5W: reconstituted with USP Normal Saline then added into 5%        Dextrose Injection bag    -   sWFI—NS: reconstituted with Sterile WFI then added into 0.9%        Sodium Chloride Injection Bag    -   NS—NS: reconstituted with USP Normal Saline then added into 0.9%        Sodium Chloride Injection bag

Example 15: Excipients in Ceftolozane Drug Product Intermediate

The excipients in exemplary ceftolozane compositionswere chosen toensure stability and processability of the ceftolozane drug substanceinto the drug product. The specific excipients, their quantities andfunctions are provided in Table 31. All excipients are compendial andtypical for sterile pharmaceutical dosage forms, requiring no additionaltreatment prior to use in the formulation. The excipients are used inlevels within the range established in other FDA approved products asdescribed in the Inactive Ingredients Database (IID).

TABLE 31 Excipients Used in Ceftolozane Composition Inactive Amount,Concentration Ingredients mg/ in Infusion Rationale for DatabaseComponent Function container Solution, % Inclusion (IID) Range Citricacid Chelating  21 0.02 Used to prevent 0.0025 to 50%  agentdiscoloration and degradation Sodium Stabilizing 487 0.49 Used as astabilizing 0.187 to 45% Chloride agent agent for ceftolozane sulfateL-arginine Alkalizing 600^((a)) 0.60 Used to adjust  0.29 to 88% agentQ.S. for pH ceftolozane solution adjustment pH ^((a))L-arginine is addedas needed to achieve pH 6.5 ± 0.5; 600 mg per container is considered arepresentative total amount.

Example 16: Manufacturing Process of a CXA-201 Composition (ComprisingTazobactam and Ceftolozane) by Co-Filling

The ceftolozane/tazobactam finished drug product is a sterile powderfill of lyophilized active ingredients ceftolozane drug productintermediate (composition) and tazobactam sodium together into a sterilesingle container. The lyophilized form of the sterile tazobactam sodiumcontains no excipients. Ceftolozane sulfate drug substance is convertedfirst into a sterile drug product intermediate, composition, byformulation with citric acid, sodium chloride and L-arginine, followedby lyophilization.

The full manufacturing process includes unit operations typical of anaseptic lyophilization process and aseptic powder filling process. Theoverall process can be outlined in two stages, as presented in themanufacturing flow chart of FIG. 12. The first stage is themanufacturing of the sterile ceftolozane composition. The second stageis the filling of the sterile drug powders into containers for the finaldrug product. The major process steps are:

Preparation of the sterile ceftolozane composition comprises

-   -   compounding the bulk solution for lyophilization;    -   sterile filtering the bulk solution;    -   lyophilizing the bulk solution into bulk powder;    -   grinding and sieving of the sterile bulk powder; and    -   aseptic packaging of the sterile bulk powder in Sterbags®.        Filling of the sterile bulk powders comprises    -   receipt of ceftolozane and tazobactam sterile powders at site;    -   aseptic filling both sterile powders into the container        sequentially;    -   blanketing the container with a nitrogen headspace;    -   stoppering and crimping the container; and    -   inspecting the container prior to secondary packaging.

What is claimed is:
 1. A method for treating an infection selected fromthe group consisting of a complicated intra-abdominal infection and acomplicated urinary tract infection in a human patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of a pharmaceutical composition comprising ceftolozane sulfateand tazobactam sodium, said composition comprising less than 0.15% of acompound of formula (III)

relative to ceftolozane sulfate, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm, when stored as asolid for 1 month at a temperature of 25° C. and at a relative humidityof 60%, wherein the ceftolozane sulfate and tazobactam sodium provideceftolozane active and tazobactam active in a ratio of 2:1 by weight. 2.The method of claim 1, wherein the infection is a complicatedintra-abdominal infection.
 3. The method of claim 1, wherein theinfection is a complicated urinary tract infection.
 4. The method ofclaim 1, wherein the composition contains less than 0.1% of the compoundof formula (III) relative to ceftolozane sulfate, as determined by highperformance liquid chromatography (HPLC) at a wavelength of 254 nm, whenstored as a solid for 1 month at a temperature of 25° C. and at arelative humidity of 60%.
 5. The method of claim 1, wherein thecomposition contains less than 0.03% of the compound of formula (III)relative to ceftolozane sulfate, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm when stored as asolid for 1 month at a temperature of 25° C. and at a relative humidityof 60%.
 6. The method of claim 1, wherein the amount of ceftolozaneactive in the pharmaceutical composition is 1,000 mg and the amount oftazobactam active in the pharmaceutical composition is 500 mg.
 7. Themethod of claim 1, wherein the amount of ceftolozane active in thepharmaceutical composition is 2,000 mg and the amount of tazobactamactive in the pharmaceutical composition is 1,000 mg.
 8. The method ofclaim 1, wherein the composition comprises 1,147 mg ceftolozane sulfateand 537 mg tazobactam sodium.
 9. The method of claim 1, wherein thecomposition is provided in a unit dosage form.
 10. The method of claim1, wherein the pharmaceutical composition is dissolved in apharmaceutically acceptable carrier and administered by parenteraladministration.
 11. The method of claim 1, wherein the pharmaceuticalcomposition is dissolved in a pharmaceutically acceptable carrier andadministered by intravenous infusion.
 12. The method of claim 2, whereinthe pharmaceutical composition is dissolved in a pharmaceuticallyacceptable carrier and administered by intravenous infusion.
 13. Themethod of claim 3, wherein the pharmaceutical composition is dissolvedin a pharmaceutically acceptable carrier and administered by intravenousinfusion.
 14. A method for treating an infection selected from the groupconsisting of a complicated intra-abdominal infection and a complicatedurinary tract infection in a human patient in need thereof comprisingadministering to the patient a therapeutically effective amount of apharmaceutical composition comprising ceftolozane sulfate and tazobactamsodium, said composition comprising less than 0.15% of the compound offormula (III)

relative to ceftolozane sulfate, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm, and less than1.5% of a compound of formula (IV)

relative to ceftolozane sulfate, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm, when stored as asolid for 3 months at a temperature of 25° C. and at a relative humidityof 60%, wherein the ceftolozane sulfate and tazobactam sodium provideceftolozane active and tazobactam active in a ratio of 2:1 by weight.15. The method of claim 14, wherein the infection is a complicatedintra-abdominal infection.
 16. The method of claim 14, wherein theinfection is a complicated urinary tract infection.
 17. The method ofclaim 14, wherein the composition contains less than 0.05% of thecompound of formula (III) relative to ceftolozane sulfate, as determinedby high performance liquid chromatography (HPLC) at a wavelength of 254nm, when stored as a solid for 3 months at a temperature of 25° C. andat a relative humidity of 60%.
 18. The method of claim 14, wherein thepharmaceutical composition is dissolved in a pharmaceutically acceptablecarrier and administered by intravenous infusion.
 19. A method fortreating an infection selected from the group consisting of acomplicated intra-abdominal infection and a complicated urinary tractinfection in a human patient in need thereof comprising administering tothe patient a therapeutically effective amount of a pharmaceuticalcomposition comprising ceftolozane sulfate and tazobactam sodium, saidcomposition comprising less than 0.15% relative to ceftolozane sulfateof the compound having a mass spectra depicted in FIG. 14, as determinedby high performance liquid chromatography (HPLC) at a wavelength of 254nm, when stored as a solid for 1 month at a temperature of 25° C. and ata relative humidity of 60%, wherein the ceftolozane sulfate andtazobactam sodium provide ceftolozane active and tazobactam active in aratio of 2:1 by weight.
 20. The method of claim 19, wherein theinfection is a complicated intra-abdominal infection.
 21. The method ofclaim 19, wherein the infection is a complicated urinary tractinfection.
 22. The method of claim 19, wherein the composition containsless than 0.1% relative to ceftolozane sulfate of the compound with themass spectra depicted in FIG. 14, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm, when stored as asolid for 1 month at a temperature of 25° C. and at a relative humidityof 60%.
 23. The method of claim 19, wherein the composition containsless than 0.03% relative to ceftolozane sulfate of the compound with themass spectra depicted in FIG. 14, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm, when stored as asolid for 1 month at a temperature of 25° C. and at a relative humidityof 60%.
 24. The method of claim 19, wherein the amount of ceftolozaneactive in the pharmaceutical composition is 1,000 mg and the amount oftazobactam active in the pharmaceutical composition is 500 mg.
 25. Themethod of claim 19, wherein the amount of ceftolozane active in thepharmaceutical composition is 2,000 mg and the amount of tazobactamactive in the pharmaceutical composition is 1,000 mg.
 26. The method ofclaim 19, wherein the composition comprises 1,147 mg ceftolozane sulfateand 537 mg tazobactam sodium.
 27. The method of claim 19, wherein thepharmaceutical composition is dissolved in a pharmaceutically acceptablecarrier and administered by intravenous infusion.
 28. A method fortreating an infection selected from the group consisting of acomplicated intra-abdominal infection and a complicated urinary tractinfection in a human patient in need thereof comprising administering tothe patient a therapeutically effective amount of a pharmaceuticalcomposition comprising ceftolozane sulfate and tazobactam sodium, saidcomposition comprising less than 0.15% relative to ceftolozane sulfateof a compound having a mass spectra depicted in FIG. 14, as determinedby high performance liquid chromatography (HPLC) at a wavelength of 254nm, and less than 1.5% of a compound of formula (IV):

relative to ceftolozane sulfate, as determined by high performanceliquid chromatography (HPLC) at a wavelength of 254 nm, when stored as asolid for 3 months at a temperature of 25° C. and at a relative humidityof 60%, wherein the ceftolozane sulfate and tazobactam sodium provideceftolozane active and tazobactam active in a ratio of 2:1 by weight.29. The method of claim 28, wherein the infection is a complicatedintra-abdominal infection.
 30. The method of claim 28, wherein theinfection is a complicated urinary tract infection.
 31. The method ofclaim 28, wherein the pharmaceutical composition is dissolved in apharmaceutically acceptable carrier and administered by intravenousinfusion.